Oligonucleotides having A-DNA form and B-DNA form conformational geometry

ABSTRACT

Modified oligonucleotides containing both A-form conformation geometry and B-from conformation geometry nucleotides are disclosed. The B-form geometry allows the oligonucleotide to serve as substrates for RNase H when bound to a target nucleic acid strand. The A-form geometry imparts properties to the oligonucleotide that modulate binding affinity and nuclease resistance. By utilizing C2′ endo sugars or O4′ endo sugars, the B-form characteristics are imparted to a portion of the oligonucleotide. The A-form characteristics are imparted via use of either 2′-O-modified nucleotides that have 3′ endo geometries or use of end caps having particular nuclease stability or by use of both of these in conjunction with each other.

CROSS-REFERENCE OF RELATED APPLICATIONS

[0001] This Application is a continuation-in-part of Ser. No.09/303,586, filed May 3, 1999 and of Ser. No. 08/936,166, filed Sep. 23,1997. Application Ser. No. 08/936,166 is a divisional of Ser. No.07/835,932, filed Mar. 5, 1992, now U.S. Pat. No. 5,670,633, whichderives from International Patent Application Serial No. PCT/US91/05720,filed Aug. 12, 1991 and published as WO 92/03568 on Mar. 5, 1992. Eachof the foregoing applications is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

[0002] The present invention relates to oligonucleotides that have bothA-form and B-form conformational geometry and methods of using sucholigonucleotides. The oligonucleotides of the invention are useful intherapeutic and investigative purposes. More specifically, the presentinvention is directed to oligonucleotides having particularmodifications that will increase affinity and nuclease resistance whileconcurrently serving as substrates for RNase H when bound to a targetRNA strand.

BACKGROUND OF THE INVENTION

[0003] It is well known that most of the bodily states in mammals,including most disease states, are affected by proteins. Classicaltherapeutic modes have generally focused on interactions with suchproteins in an effort to moderate their disease-causing or disease-potentiating functions. However, recently, attempts have been made tomoderate the actual production of such proteins by interactions withmolecules that direct their synthesis, such as intracellular RNA. Byinterfering with the production of proteins, maximum therapeutic effectand minimal side effects may be realized. It is the general object ofsuch therapeutic approaches to interfere with or otherwise modulate geneexpression leading to undesired protein formation.

[0004] One method for inhibiting specific gene expression is the use ofoligonucleotides. Oligonucleotides are now accepted as therapeuticagents. A first such oligonucleotide has been approved for humantherapeutic use by the FDA and is available in commercial marketplace.

[0005] Oligonucleotides are known to hybridize to single-stranded DNA orRNA molecules. Hybridization is the sequence-specific base pair hydrogenbonding of nucleobases of the oligonucleotide to the nucleobases of thetarget DNA or RNA molecule. Such nucleobase pairs are said to becomplementary to one another. The concept of inhibiting gene expressionthrough the use of sequence-specific binding of oligonucleotides totarget RNA sequences, also known as antisense inhibition, has beendemonstrated in a variety of systems, including living cells (forexample see: Wagner et al., Science (1993) 260: 1510-1513; Milligan etal., J. Med. Chem., (1993) 36:1923-37; Uhlmann et al., Chem. Reviews,(1990) 90:543-584; Stein et al., Cancer Res., (1988) 48:2659-2668).

[0006] The events that provide the disruption of the nucleic acidfunction by antisense oligonucleotides (Cohen in Oligonucleotides:Antisense Inhibitors of Gene Expression, (1989) CRC Press, Inc., BocaRaton, Fla.) are thought to be of two types. The first, hybridizationarrest, denotes the terminating event in which the oligonucleotideinhibitor binds to the target nucleic acid and thus prevents, by simplesteric hindrance, the binding of essential proteins, most oftenribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides:Miller, P. S. and Ts'O, P. O. P. (1987) Anti-Cancer Drug Design,2:117-128, and α-anomer oligonucleotides are the two most extensivelystudied antisense agents which are thought to disrupt nucleic acidfunction by hybridization arrest.

[0007] The second type of terminating event for antisenseoligonucleotides involves the enzymatic cleavage of the targeted RNA byintracellular RNase H. A 2′-deoxyribofuranosyl oligonucleotide oroligonucleotide analog hybridizes with the targeted RNA and this duplexactivates the RNase H enzyme to cleave the RNA strand, thus destroyingthe normal function of the RNA. Phosphorothioate oligonucleotides areprobably the most prominent example of an antisense agent that operatesby this type of antisense terminating event.

[0008] Oligonucleotides may also bind to duplex nucleic acids to formtriplex complexes in a sequence specific manner via Hoogsteen basepairing (Beal et al., Science, (1991)251:1360-1363; Young et al., Proc.Natl. Acad. Sci. (1991) 88:10023-10026). Both antisense and triple helixtherapeutic strategies are directed towards nucleic acid sequences thatare involved in or responsible for establishing or maintaining diseaseconditions. Such target nucleic acid sequences may be found in thegenomes of pathogenic organisms including bacteria, yeasts, fungi,protozoa, parasites, viruses, or may be endogenous in nature. Byhybridizing to and modifying the expression of a gene important for theestablishment, maintenance or elimination of a disease condition, thecorresponding condition may be cured, prevented or ameliorated.

[0009] In determining the extent of hybridization of an oligonucleotideto a complementary nucleic acid, the relative ability of anoligonucleotide to bind to the complementary nucleic acid may becompared by determining the melting temperature of a particularhybridization complex. The melting temperature (T_(m)), a characteristicphysical property of double helices, denotes the temperature (in degreescentigrade) at which 50% helical (hybridized) versus coil (unhybridized)forms are present. T_(m) is measured by using the UV spectrum todetermine the formation and breakdown (melting) of the hybridizationcomplex. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands.

[0010] Oligonucleotides may also be of therapeutic value when they bindto non-nucleic acid biomolecules such as intracellular or extracellularpolypeptides, proteins, or enzymes. Such oligonucleotides are oftenreferred to as ‘aptamers’ and they typically bind to and interfere withthe function of protein targets (Griffin, et al., Blood, (1993),81:3271-3276; Bock, et al., Nature, (1992) 355: 564-566).

[0011] Oligonucleotides and their analogs have been developed and usedfor diagnostic purposes, therapeutic applications and as researchreagents. For use as therapeutics, oligonucleotides must be transportedacross cell membranes or be taken up by cells, and appropriatelyhybridize to target DNA or RNA. These critical functions depend on theinitial stability of the oligonucleotides toward nuclease degradation. Aserious deficiency of unmodified oligonucleotides which affects theirhybridization potential with target DNA or RNA for therapeutic purposesis the enzymatic degradation of administered oligonucleotides by avariety of intracellular and extracellular ubiquitous nucleolyticenzymes referred to as nucleases. For oligonucleotides to be useful astherapeutics or diagnostics, the oligonucleotides should demonstrateenhanced binding affinity to complementary target nucleic acids, andpreferably be reasonably stable to nucleases and resist degradation. Fora non-cellular use such as a research reagent, oligonucleotides need notnecessarily possess nuclease stability.

[0012] A number of chemical modifications have been introduced intooligonucleotides to increase their binding affinity to target DNA or RNAand resist nuclease degradation.

[0013] Modifications have been made to the ribose phosphate backbone toincrease the resistance to nucleases. These modifications include use oflinkages such as methyl phosphonates, phosphorothioates andphosphorodithioates, and the use of modified sugar moieties such as2′-O-alkyl ribose. Other oligonucleotide modifications include thosemade to modulate uptake and cellular distribution. A number ofmodifications that dramatically alter the nature of the internucleotidelinkage have also been reported in the literature. These includenon-phosphorus linkages, peptide nucleic acids (PNA's) and 2′-5′linkages. Another modification to oligonucleotides, usually fordiagnostic and research applications, is labeling with non-isotopiclabels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, orother reporter molecules.

[0014] A variety of modified phosphorus-containing linkages have beenstudied as replacements for the natural, readily cleaved phosphodiesterlinkage in oligonucleotides. In general, most of them (such as thephosphorothioate, phosphoramidates, phosphonates andphosphorodithioates) result in oligonucleotides with reduced binding tocomplementary targets and decreased hybrid stability. At least one dozenphosphorothioate oligonucleotides and derivatives are presently beingused as antisense agents in human clinical trials for the treatment ofvarious disease states. The antisense drug Vitravin™, for use to treatcytomegalovirus (CMV) retinitis in humans, has been approved byregulatory agencies and is comedically marketed.

[0015] The structure and stability of chemically modified nucleic acidsis of great importance to the design of antisense oligonucleotides. Overthe last ten years, a variety of synthetic modifications have beenproposed to increase nuclease resistance, or to enhance the affinity ofthe antisense strand for its target mRNA (Crooke et al., Med. Res. Rev.,1996, 16, 319-344; De Mesmaeker et al., Acc. Chem. Res., 1995, 28,366-374).

[0016] RNA exists in what has been termed “A Form” geometry, while DNAexists in “B Form” geometry. In general, RNA:RNA duplexes are morestable, or have higher melting temperatures (Tm) than DNA:DNA duplexes(Sanger et al., Principles of Nucleic Acid Structure, 1984,Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34,10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). Theincreased stability of RNA has been attributed to several structuralfeatures, most notably the improved base stacking interactions thatresult from an A-form geometry (Searle et al., Nucleic Acids Res., 1993,21, 2051-2056). The presence of a hydroxyl group in the2′-pentofuranosyl (i.e., 2′-sugar) position in RNA is believed to biasthe sugar toward a C3′ endo pucker (also known as a Northern pucker),which causes the duplex to favor the A-form geometry. On the other hand,2′-deoxy nucleic acids (those having 2′-deoxy-erythro-pentofuranosylnucleotides) prefer a C2′ endo sugar pucker (also known as Southernpucker), which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). In addition, the 2′ hydroxyl groups ofRNA can form a network of water mediated hydrogen bonds that helpstabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35,8489-8494).

[0017] DNA:RNA hybrid duplexes are usually less stable than pure RNA:RNAduplexes, and depending on their sequence may be either more or lessstable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993,21, 2051-2056). The structure of a hybrid duplex is intermediate betweenA- and B-form geometries, which may result in poor stacking interactions(Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroffet al., J.Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34,4969-4982; Hortonetal., J. Mol. Biol., 1996, 264, 521-533). Thestability of a DNA:RNA hybrid is central to antisense therapies as themechanism requires the binding of a modified DNA strand to a mRNAstrand. To effectively inhibit the mRNA, the antisense DNA should have avery high binding affinity with the mRNA. Otherwise the desiredinteraction between the DNA and target mRNA strand will occurinfrequently, thereby decreasing the efficacy of the antisenseoligonucleotide.

[0018] One synthetic 2′-modification that imparts increased nucleaseresistance and a very high binding affinity to nucleotides is the2′-methoxyethoxy (MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J.Biol. Chem., 1997, 272, 11944-12000; Freier et al., Nucleic Acids Res.,1997, 25, 4429-4443). One of the immediate advantages of the MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl (Freier and Altmann, Nucleic Acids Research, (1997)25:4429-4443). 2′-O-Methoxyethyl-substituted compounds also have beenshown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Such compounds typically displayimproved RNA affinity and higher nuclease resistance relative to DNA.Chimeric oligonucleotides with 2′-O-methoxyethyl-ribonucleoside wingsand a central DNA-phosphorothioate window also have been shown toeffectively reduce the growth of tumors in animal models at low doses.MOE substituted oligonucleotides have shown outstanding promise asantisense agents in several disease states. One such MOE substitutedoligonucleotide is presently being investigated in clinical trials forthe treatment of CMV retinitis.

[0019] Recently Damha et. al., published two paper describing certainoligonucleotides that utilized arabino-pentofuranosyl nucleotides asbuilding blocks (Damha et. al., J.A.C.S., 1998, 120, 12976-12977 andDamha et. al., Bioconjugate Chem., 1999, 10, 299-305). Thearabino-pentofuranosyl oligonucleotides, i.e., arabinonucleic acids,described by Damha et. al., utilized either arabinose or2′-deoxy-2′-fluoro arabinose as the sugar unit of their respectivenucleotides. In one of the two arabinonucleic acids described, all ofthe nucleotides of the nucleic acid were arabinose and in the other, allof the nucleotides were 2′-deoxy-2′-fluoro arabinose. In both of thesenucleic acids, the nucleotides were joined via phosphodiester linkages.These authors were able to show that the 2′-fluoro arabino-containingoligonucleotides when bound to RNA activated cleavage of the RNA by E.coli and HIV-RT RNase H. The authors further noted that while the twoarabinonucleic acids they described were more stable to serum andcellular nucleases than DNA they were less stable than normalphosphorothioate deoxyoligonucleotides.

[0020] Although the known modifications to oligonucleotides havecontributed to the development of oligonuclotides for various uses,including use in diagnostics, therapeutics and as research reagents,there still exists a need in the art for further oligonucleotides havingenhanced hybrid binding affinity and/or increased nuclease resistanceand that can take advantage of the RNase H termination mechanism.

SUMMARY OF THE INVENTION

[0021] In one aspect, the present invention is directed tooligonucleotides having multiple properties. One of these properties isthe ability to form a double stranded structure with an RNA and elicitRNase H cleavage of the RNA. Further properties of the oligonucleotidesinclude having improved binding affinity and nuclease resistance. Theoligonucleotides of the invention comprise oligonucleotide formed from aplurality of nucleotides. A first portion of the nucleotides are joinedtogether in a contiguous sequence with each nucleotide of this portionselected as a nucleotide that has B-form conformational geometry whenjoined in a contiguous sequence with other nucleotides. Included in thisfirst portion of nucleotides are ribonucleotides or arabino nucleotides.The oligonucleotides include a further portion of nucleotides that arejoined together in at least one contiguous sequence. Each of thesefurther nucleotides are selected as ribonucleotides that have A-formconformational geometry when joined in a contiguous sequence.

[0022] In preferred embodiments of the invention, each of thenucleotides of the first portion of nucleotides, independently, areselected to be 2′-SCH₃ ribonucleotides, 2′-NH₂ ribonucleotides,2′-NH(C₁-C₂ alkyl) ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ ribonucleotides,2′-CF₃ ribonucleotides, 2′=CH₂ ribonucleotides, 2′=CHF ribonucleotides,2′=CF₂ ribonucleotides, 2′-CH₃ ribonucleotides. 2′-C₂H₅ ribonucleotides,2′-CH═CH₂ ribonucleotides or 2′-C≡CH ribonucleotides. These are joinedtogether in a contiguous sequence by phosphate, phosphorothioate,phosphorodithioate or boranophosphate linkages.

[0023] In a further preferred embodiment of the invention, each of thenucleotides of said further portion of nucleotides, independently, areselected to be 2′-fluoro nucleotides or nucleotides having a2′-substituent having the formula I or II:

[0024] wherein

[0025] E is C₁-C₁₀ alkyl, N(Q₁)(Q₂) or N═C(Q₁,)(Q₂);

[0026] each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl,dialkylaminoalkyl, a nitrogen protecting group, a tethered or untetheredconjugate group, a linker to a solid support, or Q₁ and Q₂, together,are joined in a nitrogen protecting group or a ring structure that caninclude at least one additional heteroatom selected from N and O;

[0027] R₃ is OX, SX, or N(X)₂;

[0028] each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)Z, C(═O)N(H)Z or OC(═O)N(H)Z;

[0029] Z is H or C₁-C₈ alkyl;

[0030] L₁, L₂ and L₃ comprise a ring system having from about 4 to about7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

[0031] Y is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q₁)(Q₂), O(Q₁),halo, S(Q₁), or CN;

[0032] each q₁ is, independently, from 2 to 10;

[0033] each q₂ is, independently, 0 or 1;

[0034] m is 0, 1 or2;

[0035] p is from 1 to 10; and

[0036] q₃ is from 1 to 10 with the proviso that when p is 0, q₃ isgreater than 1.

[0037] A more preferred group for use as the further portion ofnucleotides are 2′-F ribonucleotides, 2′-O-(C₁-C₆ alkyl)ribonucleotides, or 2′-O-(C₁-C₆ substituted alkyl) ribonucleotideswherein the substitution is C₁-C₆ ether, C₁-C₆ thioether,amino,amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂. These nucleotides arejoined together in sequence by 3′-5′ phosphodiester, 2′-5′phosphodiester, phosphorothioate, Sp phosphorothioate, Rpphosphorothioate, phosphorodithioate, 3′-deoxy-3′-aminophosphoroarnidate, 3′-methylenephosphonate, methylene(methylimino),dimethylhydazino, amide 3 (i.e., (3′)—CH₂—NH—C(O)—(5′)), amide 4 (i.e.,(3′)—CH₂—C(O)—NH—(5′) boranophosphate linkages.

[0038] In one preferred embodiment of the invention, at least two of thenucleotides of the further portion of nucleotides are joined together ina contiguous sequence that is position 3′ to the contiguous sequence ofthe first portion of nucleotides. In an additional preferred embodimentof the invention, at least two of the further portion of nucleotides arejoined together in a continuous sequence that is position 5′ to thecontinuous sequence of the first portion of nucleotides.

[0039] In a further preferred embodiment of the invention, at least twoof the nucleotides of the further portion of nucleotides are joinedtogether in a continuous sequence that is position 3′ to the continuoussequence of the first portion of nucleotides and at least two of thefurther portion of nucleotides are joined together in a continuoussequence that is position 5′ to the continuous sequence of the firstportion of nucleotides.

[0040] A first preferred group of nucleotides for use as the firstportion of nucleotides include 2′-SCH₃ ribonucleotides, 2′-NH2ribonucleotides, 2′-NH(C₁-C₂ alkyl) ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ribonucleotides, 2′=CH₂ ribonucleotides, 2′-CH₃ ribonucleotides, 2′-C₂H₅ribonucleotides, 2′-CH═CH₂ ribonucleotides and 2′-C≡CH ribonucleotides.A more preferred group include 2′-SCH₃ ribonucleotides, 2′-NH₂ribonucleotides, 2′-NH(C₁-C₂ alkyl) ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ribonucleotides and 2′-CH₃ ribonucleotides. A further preferred groupinclude 2′-SCH₃ ribonucleotides, 2′-NH₂ribonucleotides and2′-CH₃ribonucleotides. Particularly preferred are is 2′-SCH₃ribonucleotides.

[0041] A further group of nucleotides that are preferred of use as thenucleotides of the first portion of the oligonucleotides of theinventions are 2′-CN arabino nucleotides, 2′-F arabino nucleotides,2′-Cl arabino nucleotides, 2′-Br arabino nucleotides, 2′-N₃ arabinonucleotides, 2′-OH arabino nucleotides, 2′-O—CH₃ arabino nucleotides and2′-dehydro-2′-CH₃ arabino nucleotides. A more preferred group include2′-F arabino nucleotides, 2′-OH arabino nucleotides and 2′-O—CH₃ arabinonucleotides. A further preferred group include 2′-F arabino nucleotidesand 2′-OH arabino nucleotides. Particularly preferred are 2′-F arabinonucleotides.

[0042] Particularly preferred oligonucleotides of the invention includeselecting the nucleotides of the first portion of nucleotides to be2′-SCH₃ ribonucleotides, 2′-NH₂ ribonucleotides, 2′-NH(C₁-C₂ alkyl)ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ ribonucleotides, 2′-CH₃ribonucleotides, 2′-CH═CH₂ ribonucleotides or 2′-C≡CH ribonucleotidesand selecting the nucleotides of the further portion of nucleotides tobe 2′-F ribonucleotides, 2′-O—(C₁-C₆ alkyl) ribonucleotides or2′-O—(C₁-C₆ substituted alkyl) ribonucleotides wherein the substitutionis C₁-C₆ ether, C₁-C₆ thioether, amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.

[0043] Further preferred oligonucleotides of the invention includeselecting the nucleotides of said first portion of nucleotides to be2′-CN arabino nucleotides, 2′-F arabino nucleotides, 2′-Cl arabinonucleotides, 2′-Br arabino nucleotides, 2′-N₃ arabino nucleotides, 2′-OHarabino nucleotides, 2′-O—CH₃ arabino nucleotides or 2′-dehydro-2′-CH₃arabino nucleotides and selecting the nucleotides of the further portionof nucleotides to be 2′-F ribonucleotides, 2′-O—(C₁-C₆ alkyl)ribonucleotides or 2′-O—(C₁-C₆ substituted alkyl) ribonucleotideswherein the substitution is C₁-C₆ ether, C₁-C₆ thioether, amino,amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.

[0044] Particularly preferred are oligonucleotide of the invention whereeach nucleotide of the first portion of nucleotides is a 2′-F arabinonucleotides or a 2′-OH arabino nucleotides and each nucleotide of thefurther portion of nucleotides is a 2′-O—(C₁-C₆ substituted alkyl)ribonucleotide wherein the substitution is C₁-C₆ ether, C₁-C₆ thioether,amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.

[0045] In further preferred oligonucleotides of the invention thefurther portion of the plurality of nucleotides comprise at least twonucleotides joined together in a contiguous sequence that is position atthe 3′ terminus end of the oligonucleotide. In an additional preferredoligonucleotide of the invention the further portion of said pluralityof nucleotides comprise at least two nucleotides joined together in acontiguous sequence that is position at the 5′ terminus end of theoligonucleotide. In even further preferred oligonucleotides of theinvention the further portion of the plurality of nucleotides compriseat least two nucleotides joined together in a contiguous sequence thatis position at the 3′ terminus end of the oligonucleotide and at leasttwo nucleotides joined together in a contiguous sequence that isposition at the 5′ terminus end of the oligonucleotide. Preferredlinkages for joining these nucleotides together in an oligonucleotide ofthe invention include 2′-5′ phosphodiester linkages,3′-methylenephosphonate linkages, Sp phosphorothioate linkages,methylene(methylimino)linkages, dimethyhydrazino linkages,3′-deoxy-3′-amino phosphoroamidate linkages, amide 3 linkages or amide 4linkages. Particularly preferred joining linkages are 2′-5′phosphodiester linkages, 3′-methylenephosphonate linkages, Spphosphorothioate linkages or methylene(methylimino) linkages.

[0046] In further preferred oligonucleotides of the invention,nucleotides for use in the further portion of nucleotides comprises2′-alkylamino substituted nucleotides located at the 3′ terminus, the 5′terminus or both the 3′ and 5′ terminus of the oligonucleotide.Particularly preferred are 2′-O-alkylamines such as 2′-O-ethylamine and2′-O-propylamine.

[0047] Further oligonucleotides of the invention compriseoligonucleotides made up of a plurality of linked nucleotides at leasttwo of which comprise nucleotides that are not2′-deoxy-erthro-pentofuranosyl nucleotides and that have a C2′ endo typepucker or an O4′ endo type pucker and that are joined together in acontiguous sequence and other nucleotides comprising nucleotides thathave a C3′ endo type pucker. Preferred are oligonucleotides having theC3′ endo type pucker nucleotides joined together in a contiguoussequence that is positioned 3′ to the contiguous sequence of thenucleotides having the C2′ endo type pucker or O4′ endo type pucker.Further preferred oligonucleotides are oligonucleotides wherein thenucleotides having the C3′ endo type pucker are joined together in acontiguous sequence that is positioned 5′ to the contiguous sequence ofhaving the C2′ endo type pucker or O4′ endo type pucker. Additionalpreferred oligonucleotide are oligonucleotides where a portion of thenucleotides having the C3′ endo type pucker are joined together in acontiguous sequence that is positioned 3′ to the contiguous sequence ofnucleotides having the C2′ endo type pucker or O4′ endo type pucker anda further portion of nucleotides having the C3′ endo type pucker arejoined together in a contiguous sequence that is positioned 5′ to thecontiguous sequence of nucleotides having the C2′ endo type pucker orO4′ endo type pucker.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1 illustrates a preferred group of nucleotide fragments foruse in the B-form portion (the C2′ endo/O4′ endo portion) ofoligonucleotides of the invention.

[0049]FIG. 2 illustrates a preferred group of nucleotide fragments foruse in the A-form portion (the C3′ endo portion) of oligonucleotides ofthe invention.

[0050]FIG. 3 illustrates a preferred group of nucleotide fragments foruse in A-form portions at the 3′ terminus of oligonucleotides of theinvention.

[0051]FIG. 4 is a plot of the percentage of full length oligonucleotideremaining intact in plasma one hour following administration of an i.v.bolus of 5 mg/kg oligonucleotide.

[0052]FIG. 5 is a plot of the percentage of full length oligonucleotideremaining intact in tissue 24 hours following administration of an i.v.bolus of 5 mg/kg oligonucleotide.

[0053]FIG. 6 shows CGE traces of test oligonucleotides and a standardphosphorothioate oligonucleotide in both mouse liver samples and mousekidney samples after 24 hours.

[0054]FIG. 7 shows a graph of the effect of the oligonucleotides of thepresent invention on c-raf expression (compared to control) in bENDcells.

[0055]FIGS. 8 and 9 shows bar graphs as percent control normalized forthe G3PDH signal eighteen hours after treatment with specifiedcompounds.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0056] In one aspect, the present invention is directed to noveloligonucleotides that have certain desirable properties that contributeto increases in binding affinity and/or nuclease resistance, coupledwith the ability to serve as substrates for RNase H.

[0057] The oligonucleotide of the invention are formed from a pluralityof nucleotides that are joined together via internucleotide linkages.While joined together as a unit in the oligonucleotide, the individualnucleotides of oligonucleotides are of several types. Each of thesetypes contribute unique properties to the oligonucleotide. A first typeof nucleotides are joined together in a continuous sequence that forms afirst portion of the oligonucleotide. The remaining nucleotides are ofat least one further type and are located in one or more remainingportions or locations within the oligonucleotide. Thus, theoligonucleotides of the invention include a nucleotide portion thatcontributes one set of attributes and a further portion (or portions)that contributes another set of attributes.

[0058] One attribute that is desirable is eliciting RNase H activity. Toelicit RNase H activity, a portion of the oligonucleotides of theinvention is selected to have B-form like conformational geometry. Thenucleotides for this B-form portion are selected to specifically includeribo-pentofuranosyl and arabino-pentofuranosyl nucleotides.2′-Deoxy-erythro-pentfuranosyl nucleotides also have B-form geometry andelicit RNase H activity. While not specifically excluded, if2′-deoxy-erythro-pentfuranosyl nucleotides are included in the B-formportion of an oligonucleotide of the invention, such2′-deoxy-erythro-pentfuranosyl nucleotides preferably does notconstitute the totality of the nucleotides of that B-form portion of theoligonucleotide, but should be used in conjunction with ribonucleotidesor an arabino nucleotides. As used herein, B-form geometry is inclusiveof both C2′-endo and O4′-endo pucker, and the ribo and arabinonucleotides selected for inclusion in the oligonucleotide B-form portionare selected to be those nucleotides having C2′-endo conformation orthose nucleotides having O4′-endo conformation. This is consistent withBerger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, whopointed out that in considering the furanose conformations in whichnucleosides and nucleotides reside, B-form consideration should also begiven to a O4′-endo pucker contribution.

[0059] A-form nucleotides are nucleotides that exhibit C3′-endo pucker,also known as north, or northern, pucker. In addition to the B-formnucleotides noted above, the A-form nucleotides can be C3′-endo puckernucleotides or can be nucleotides that are located at the 3′ terminus,at the 5′ terminus, or at both the 3′ and the 5′ terminus of theoligonucleotide. Alternatively, A-form nucleotides can exist both in aC3′-endo pucker and be located at the ends, or termini, of theoligonucleotide. In selecting nucleotides that have C3′-endo pucker orin selecting nucleotides to reside at the 3′ or 5′ ends of theoligonucleotide, consideration is given to binding affinity and nucleaseresistance properties that such nucleotides need to impart to theresulting the oligonucleotide.

[0060] Nucleotides selected to reside at the 3′ or 5′ termini ofoligonucleotides of the invention are selected to impart nucleaseresistance to the oligonucleotide. This nuclease resistance can also beachieved via several mechanisms, including modifications of the sugarportions of the nucleotide units of the oligonucleotides, modificationof the internucleotide linkages or both modification of the sugar andthe internucleotide linkage.

[0061] A particularly useful group of nucleotides for use in increasingnuclease resistance at the termini of oligonucleotides are those having2′-O-alkylamino groups thereon. The amino groups of such nucleotides canbe groups that are protonated at physiological pH. These include amines,monoalkyl substituted amines, dialkyl substituted amines andheterocyclic amines such as imidazole. Particularly useful are the loweralkyl amines including 2′-O-ethylamine and 2′-O-propylamine. SuchO-alkylamines can also be included on the 3′ position of the 3′ terminusnucleotide. Thus the 3′ terminus nucleotide could include both a 2′ anda 3′-O-alkylamino substituent.

[0062] In selecting for nuclease resistance, it is important not todetract from binding affinity. Certain phosphorus based linkage havebeen shown to increase nuclease resistance. The above describedphosphorothioate linkage increase nuclease resistance, however, it alsocauses loss of binding affinity. Thus, generally for use in thisinvention, if phosphorothioate internucleotide linkage are used, othermodification will be made to nucleotide units that increase bindingaffinity to compensate for the decreased affinity contribute by thephosphorothioate linkages.

[0063] Other phosphorus based linkages having increase nucleaseresistance that do not detract from binding affinity include3′-methylene phosphonates and 3′-deoxy-3′-amino-phosphoroamidatelinkages. A further class of linkages that contribute nucleaseresistance but do not detract from binding affinity are non-phosphate innature. Preferred among these are methylene(methylimino) linkages,dimethylhydraxino linkages, and amine 3 and amide 4 linkages asdescribed (Freier and Altmann, Nucleic Acid Research, 1997, 25,4429-4443).

[0064] There are a number of potential items to consider when designingoligonucleotides having improved binding affinities. It appears that oneeffective approach to constructing modified oligonucleotides with veryhigh RNA binding affinity is the combination of two or more differenttypes of modifications, each of which contributes favorably to variousfactors that might be important for binding affinity.

[0065] Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443,recently published a study on the influence of structural modificationsof oligonucleotides on the stability of their duplexes with target RNA.In this study, the authors reviewed a series of oligonucleotidescontaining more than 200 different modifications that had beensynthesized and assessed for their hybridization affinity and T_(m).Sugar modifications studied included substitutions on the 2′-position ofthe sugar, 3′-substitution, replacement of the 4′-oxygen, the use ofbicyclic sugars, and four member ring replacements. Several nucleobasemodifications were also studied including substitutions at the 5, or 6position of thymine, modifications of pyrimidine heterocycle andmodifications of the purine heterocycle. Numerous backbone modificationswere also investigated including backbones bearing phosphorus, backbonesthat did not bear a phosphorus atom, and backbones that were neutral.

[0066] Four general approaches might be used to improve hybridization ofoligonucleotides to RNA targets. These include: preorganization of thesugars and phosphates of the oligodeoxynucleotide strand intoconformations favorable for hybrid formation, improving stacking ofnucleobases by the addition of polarizable groups to the heterocyclebases of the nucleotides of the oligonucleotide, increasing the numberof H-bonds available for A-U pairing, and neutralization of backbonecharge to facilitate removing undesirable repulsive interactions. Wehave found that by utilizing the first of these, preorganization of thesugars and phosphates of the oligodeoxynucleotide strand intoconformations favorable for hybrid formation, to be a preferred methodto achieve improve binding affinity. It can further be used incombination with the other three approaches.

[0067] Sugars in DNA:RNA hybrid duplexes frequently adopt a C3′ endoconformation. Thus modifications that shift the conformationalequilibrium of the sugar moieties in the single strand toward thisconformation should preorganize the antisense strand for binding to RNA.Of the several sugar modifications that have been reported and studiedin the literature, the incorporation of electronegative substituentssuch as 2′-fluoro or 2′-alkoxy shift the sugar conformation towards the3′ endo (northern) pucker conformation. This preorganizes anoligonucleotide that incorporates such modifications to have an A-formconformational geometry. This A-form conformation results in increasedbinding affinity of the oligonucleotide to a target RNA strand.

[0068] Representative 2′-substituent groups amenable to the presentinvention that give A-form conformational properties to the nucleotidesinclude 2′-O-alkyl, 2′-O-substituted alkyl and 2′-fluoro substituentgroups. Preferred for the substituent groups are various alkyl and arylethers and thioethers, amines and monoalkyl and dialkyl substitutedamines. A particular preferred group include those having the formula Ior II:

[0069] wherein

[0070] E is C₁-C₁₀ alkyl, N(Q₁)(Q₂) or N═C(Q₁)(Q2);

[0071] each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl,dialkylaminoalkyl, a nitrogen protecting group, a tethered or untetheredconjugate group, a linker to a solid support, or Q₁ and Q₂, together,are joined in a nitrogen protecting group or a ring structure that caninclude at least one additional heteroatom selected from N and O;

[0072] R₃ is OX, SX, or N(X)₂;

[0073] each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)Z, C(═O)N(H)Z or OC(═O)N(H)Z;

[0074] Z is H or C₁-C8 alkyl;

[0075] L₁, L₂ and L₃ comprise a ring system having from about 4 to about7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

[0076] Y is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q₁)(Q₂), O(Q₁),halo, S(Q₁), or CN;

[0077] each q₁ is, independently, from 2 to 10;

[0078] each q₂ is, independently, 0 or 1;

[0079] m is 0, 1 or 2;

[0080] p is from 1 to 10; and

[0081] q₃ is from 1 to 10 with the proviso that when p is 0, q₃ isgreater than 1.

[0082] The above 2′-substituents confer a 3′-endo pucker to the sugarwhere they are incorporated. This pucker conformation further assists inincreasing the Tm of the oligonucleotide with its target.

[0083] The high binding affinity resulting from 2′ substitution has beenpartially attributed to the 2′ substitution causing a C3′ endo sugarpucker which in turn may give the oligomer a favorable A-form likegeometry. This is a reasonable hypothesis since substitution at the 2′position by a variety of electronegative groups (such as fluoro andO-alkyl chains) has been demonstrated to cause C3′ endo sugar puckering(De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374; Lesnik et al.,Biochemistry, 1993, 32, 7832-7838).

[0084] In addition, for 2′-substituents containing an ethylene glycolmotif, a gauche interaction between the oxygen atoms around the O—C—C—Otorsion of the side chain may have a stabilizing effect on the duplex(Freier et al., Nucleic Acids Research, (1997) 25:4429-4442). Suchgauche interactions have been observed experimentally for a number ofyears (Wolfe et al., Acc. Chem. Res., 1972, 5, 102; Abe et al., J. Am.Chem. Soc., 1976, 98, 468). This gauche effect may result in aconfiguration of the side chain that is favorable for duplex formation.The exact nature of this stabilizing configuration has not yet beenexplained. While we do not want to be bound by theory, it may be thatholding the O—C—C—O torsion in a single gauche configuration, ratherthan a more random distribution seen in an alkyl side chain, provides anentropic advantage for duplex formation.

[0085] To better understand the higher RNA affinity of 2′-O-methoxyethylsubstituted RNA and to examine the conformational properties of the2′-O-methoxyethyl substituent, a self-complementary dodecameroligonucleotide 2′-O-MOE r(CGCGAAUUCGCG) SEQ ID NO: 1 was synthesized,crystallized and its structure at a resolution of 1.7 Angstrom wasdetermined. The crystallization conditions used were 2 mMoligonucleotide, 50 mM Na Hepes pH 6.2-7.5, 10.50 mM MgCl₂, 15% PEG 400.The crystal data showed: space group C2, cell constants α=41.2 Å, b=34.4Å, c=46.6 Å, β=92.4°. The resolution was 1.7 Å at −170° C. The currentR=factor was 20% (R_(free) 26%).

[0086] This crystal structure is believed to be the first crystalstructure of a fully modified RNA oligonucleotide analogue. The duplexadopts an overall A-form conformation and all modified sugars displayC3′-endo pucker. In most of the 2′-O-substituents, the torsion anglearound the A′—B′ as depicted in Structure II below, of the ethyleneglycol linker has a gauche conformation. For 2′-O-MOE, A′ and B′ ofStructure II below are methylene moieties of the ethyl portion of theMOE and R′ is the methoxy portion.

[0087] In the crystal, the 2′-O-MOE RNA duplex adopts a generalorientation such that the crystallographic 2-fold rotation axis does notcoincide with the molecular 2-fold rotation axis. The duplex adopts theexpected A-type geometry and all of the 24 2′-O-MOE substituents werevisible in the electron density maps at full resolution. The electrondensity maps as well as the temperature factors of substituent atomsindicate flexibility of the 2′-O-MOE substituent in some cases.

[0088] Most of the 2-O-MOE substituents display a gauche conformationaround the C—C bond of the ethyl linker. However, in two cases, a transconformation around the C—C bond is observed. The lattice interactionsin the crystal include packing of duplexes against each other via theirminor grooves. Therefore, for some residues, the conformation of the2′-O-substituent is affected by contacts to an adjacent duplex. Ingeneral, variations in the conformation of the substituents (e.g. g⁺ org⁻ around the C—C bonds) create a range of interactions betweensubstituents, both inter-strand, across the minor groove, andintra-strand. At one location, atoms of substituents from two residuesare in van der Waals contact across the minor groove. Similarly, a closecontact occurs between atoms of substituents from two adjacentintra-strand residues.

[0089] Previously determined crystal structures of A-DNA duplexes werefor those that incorporated isolated 2′-O-methyl T residues. In thecrystal structure noted above for the 2′-O-MOE substituents, a conservedhydration pattern has been observed for the 2′-O-MOE residues. A singlewater molecule is seen located between O2′, O3′ and the methoxy oxygenatom of the substituent, forming contacts to all three of between 2.9and 3.4 Å. In addition, oxygen atoms of substituents are involved inseveral other hydrogen bonding contacts. For example, the methoxy oxygenatom of a particular 2′-O-substituent forms a hydrogen bond to N3 of anadenosine from the opposite strand via a bridging water molecule.

[0090] In several cases a water molecule is trapped between the oxygenatoms O2′, O3′ and OC′ of modified nucleosides. 2′-O-MOE substituentswith trans conformation around the C—C bond of the ethylene glycollinker are associated with close contacts between OC′ and N2 of aguanosine from the opposite strand, and, water-mediated, between OC′ andN3(G). When combined with the available thermodynamic data for duplexescontaining 2′-O-MOE modified strands, this crystal structure allows forfurther detailed structure-stability analysis of other antisensemodifications.

[0091] In extending the crystallographic structure studies, molecularmodeling experiments were performed to study further enhanced bindingaffinity of oligonucleotides having 2′-O-modifications of the invention.The computer simulations were conducted on compounds of SEQ ID NO: 1,above, having 2′-O-modifications of the invention located at each of thenucleoside of the oligonucleotide. The simulations were performed withthe oligonucleotide in aqueous solution using the AMBER force fieldmethod (Cornell et al., J. Am. Chem. Soc., 1995,117, 5179-5197)(modelingsoftware package from UCSF, San Francisco, Calif.). The calculationswere performed on an Indigo2 SGI machine (Silicon Graphics, MountainView, Calif.).

[0092] Further 2′-O-modifications of the inventions include those havinga ring structure that incorporates a two atom portion corresponding tothe A′ and B′ atoms of Structure II. The ring structure is attached atthe 2′ position of a sugar moiety of one or more nucleosides that areincorporated into an oligonucleotide. The 2′-oxygen of the nucleosidelinks to a carbon atom corresponding to the A′ atom of Structure II.These ring structures can be aliphatic, unsaturated aliphatic, aromaticor heterocyclic. A further atom of the ring (corresponding to the B′atom of Structure II), bears a further oxygen atom, or a sulfur ornitrogen atom. This oxygen, sulfur or nitrogen atom is bonded to one ormore hydrogen atoms, alkyl moieties, or haloalkyl moieties, or is partof a further chemical moiety such as a ureido, carbamate, amide oramidine moiety. The remainder of the ring structure restricts rotationabout the bond joining these two ring atoms. This assists in positioningthe “further oxygen, sulfur or nitrogen atom” (part of the R position asdescribed above) such that the further atom can be located in closeproximity to the 3′-oxygen atom (O3′) of the nucleoside.

[0093] The ring structure can be further modified with a group usefulfor modifying the hydrophilic and hydrophobic properties of the ring towhich it is attached and thus the properties of an oligonucleotide thatincludes the 2′-O-modifications of the invention. Further groups can beselected as groups capable of assuming a charged structure, e.g. anamine. This is particularly useful in modifying the overall charge of anoligonucleotide that includes a 2′-O-modifications of the invention.When an oligonucleotide is linked by charged phosphate groups, e.g.phosphorothioate or phosphodiester linkages, location of a counter ionon the 2′-O-modification, e.g. an amine functionality, locallynaturalizes the charge in the local environment of the nucleotidebearing the 2′-O-modification. Such neutralization of charge willmodulate uptake, cell localization and other pharmacokinetic andpharmacodynamic effects of the oligonucleotide.

[0094] Preferred ring structures of the invention for inclusion as a2′-O modification include cyclohexyl, cyclopentyl and phenyl rings aswell as heterocyclic rings having spacial footprints similar tocyclohexyl, cyclopentyl and phenyl rings. Particularly preferred2′-O-substituent groups of the invention are listed below including anabbreviation for each:

[0095] 2′-O-(trans 2-methoxy cyclohexyl)-2′-O-(TMCHL)

[0096] 2′-O-(trans 2-methoxy cyclopentyl)-2′-O-(TMCPL)

[0097] 2′-O-(trans 2-ureido cyclohexyl)-2′-O-(TUCHL)

[0098] 2′-O-(trans 2-methoxyphenyl)-2′-O-(2MP)

[0099] Structural details for duplexes incorporating such2-O-substituents were analyzed using the described AMBER force fieldprogram on the Indigo2 SGI machine. The simulated structure maintained astable A-form geometry throughout the duration of the simulation. Thepresence of the 2′ substitutions locked the sugars in the C3′-endoconformation.

[0100] The simulation for the TMCHL modification revealed that the2′-O-(TMCHL) side chains have a direct interaction with water moleculessolvating the duplex. The oxygen atoms in the 2-O-(TMCHL) side chain arecapable of forming a water-mediated interaction with the 3′ oxygen ofthe phosphate backbone. The presence of the two oxygen atoms in the2′-O-(TMCHL) side chain gives rise to favorable gauche interactions. Thebarrier for rotation around the O—C—C—O torsion is made even larger bythis novel modification. The preferential preorganization in an A-typegeometry increases the binding affinity of the 2′-O-(TMCHL) to thetarget RNA. The locked side chain conformation in the 2′-O-(TMCHL) groupcreated a more favorable pocket for binding water molecules. Thepresence of these water molecules played a key role in holding the sidechains in the preferable gauche conformation. While not wishing to bebound by theory, the bulk of the substituent, the diequatorialorientation of the substituents in the cyclohexane ring, the water ofhydration and the potential for trapping of metal ions in theconformation generated will additionally contribute to improved bindingaffinity and nuclease resistance of oligonucleotides incorporatingnucleosides having this 2′-O-modification.

[0101] As described for the TMCHL modification above, identical computersimulations of the 2′-O-(TMCPL), the 2′-O-(2MP) and 2′-O-(TUCHL)modified oligonucleotides in aqueous solution also illustrate thatstable A-form geometry will be maintained throughout the duration of thesimulation. The presence of the 2′ substitution will lock the sugars inthe C3′-endo conformation and the side chains will have directinteraction with water molecules solvating the duplex. The oxygen atomsin the respective side chains are capable of forming a water-mediatedinteraction with the 3′ oxygen of the phosphate backbone. The presenceof the two oxygen atoms in the respective side chains give rise to thefavorable gauche interactions. The barrier for rotation around therespective O—C—C—O torsions will be made even larger by respectivemodification. The preferential preorganization in A-type geometry willincrease the binding affinity of the respective 2′-O-modifiedoligonucleotides to the target RNA. The locked side chain conformationin the respective modifications will create a more favorable pocket forbinding watermolecules. The presence of these water molecules plays akey role in holding the side chains in the preferable gaucheconformation. The bulk of the substituent, the diequatorial orientationof the substituents in their respective rings, the water of hydrationand the potential trapping of metal ions in the conformation generatedwill all contribute to improved binding affinity and nuclease resistanceof oligonucleotides incorporating nucleosides having these respective2′-O-modification.

[0102] Preferred for use as the B-form nucleotides for eliciting RNase Hare ribonucleotides having 2-deoxy-2′-S-methyl, 2′-deoxy-2′-methyl,2′-deoxy-2′-amino, 2′-deoxy-2′-mono or dialkyl substituted amino,2′-deoxy-2′-fluoromethyl, 2′-deoxy-2′-difluoromethyl,2′-deoxy-2′-trifluoromethyl, 2′-deoxy-2′-methylene,2′-deoxy-2′-fluoromethylene, 2′-deoxy-2′-difluoromethylene,2′-deoxy-2′-ethyl, 2′-deoxy-2′-ethylene and 2′-deoxy-2′-acetylene. Thesenucleotides can alternately be described as 2-SCH₃ ribonucleotide,2′-CH₃ribonucleotide, 2′-NH₂ ribonucleotide 2′-NH(C₁-C₂ alkyl)ribonucleotide, 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, 2′-CH₂Fribonucleotide, 2′-CHF₂ ribonucleotide, 2′-CF₃ ribonucleotide, 2′=CH₂ribonucleotide, 2′=CHF ribonucleotide, 2′=CF₂ ribonucleotide, 2′-C₂H₅ribonucleotide, 2′-CH═CH₂ ribonucleotide, 2′-C≡CH ribonucleotide. Afurther useful ribonucleotide is one having a ring located on the ribosering in a cage-like structure including3′,O,4′-C-methyleneribonucleotides. Such cage-like structures willphysically fix the ribose ring in the desired conformation.

[0103] Additionally, preferred for use as the B-form nucleotides foreliciting RNase H are arabino nucleotides having 2′-deoxy-2′-cyano,2′-deoxy-2′-fluoro, 2′-deoxy-2′-chloro, 2′-deoxy-2′-bromo,2′-deoxy-2′-azido, 2′-methoxy and the unmodified arabino nucleotide(that includes a 2′-OH projecting upwards towards the base of thenucleotide). These arabino nucleotides can alternately be described as2′-CN arabino nucleotide, 2′-F arabino nucleotide, 2′-Cl arabinonucleotide, 2′-Br arabino nucleotide, 2′-N₃ arabino nucleotide, 2′-O—CH₃arabino nucleotide and arabino nucleotide.

[0104] Such nucleotides are linked together via phosphorothioate,phosphorodithioate, boranophosphate or phosphodiester linkages.particularly preferred is the phosphorothioate linkage.

[0105] Illustrative of the B-form nucleotides for use in the inventionis a 2′-S-methyl (2′-SMe) nucleotide that resides in C2′ endoconformation. It can be compared to 2′-O-methyl (2′-OMe) nucleotidesthat resides in a C3′ endo conformation. Particularly suitable for usein comparing these two nucleotides are molecular dynamic investigationsusing a SGI [Silicon Graphics, Mountain View, Calif.] computer and theAMBER [UCSF, San Francisco, Calif.] modeling software package forcomputer simulations.

[0106] Ribose conformations in C2′-modified nucleosides containingS-methyl groups were examined. To understand the influence of2′-O-methyl and 2′-S-methyl groups on the conformation of nucleosides, weevaluated the relative energies of the 2′-O— and 2 ′-S-methylguanosine,along with normal deoxyguanosine and riboguanosine, starting from bothC2′-endo and C3′-endo conformations using ab initio quantum mechanicalcalculations. All the structures were fully optimized at HF/6-31G* leveland single point energies with electron-correlation were obtained at theMP2/6-31G*//HF/6-31G* level. As shown in Table 1, the C2′-endoconformation of deoxyguanosine is estimated to be 0.6 kcal/mol morestable than the C3′-endo conformation in the gas-phase. Theconformational preference of the C2′-endo over the C3′-endo conformationappears to be less dependent upon electron correlation as revealed bythe MP2/6-31G*//HF/6-31G* values which also predict the same differencein energy. The opposite trend is noted for riboguanosine. At theHF/6-31G* and MP2/6-31G*//HF/6-31G* levels, the C3′-endo form ofriboguanosine is shown to be about 0.65 and 1.41 kcal/mol more stablethan the C2′ endo form, respectively. TABLE 1 Relative energies* of theC3'-endo and C2'-endo confonnations of representative nucleosides.CONTINUUM HF/6-31GMP2/6-31-G MODEL AMBER dG 0.60 0.56 0.88 0.65 rG −0.65−1.41 −0.28 −2.09 2'-O-MeG −0.89 −1.79 −0.36 −0.86 2'-S-MeG 2.55 1.413.16 2.43

[0107] Table 1 also includes the relative energies of2′-O-methylguanosine and 2′-S-methylguanosine in C2′-endo and C3′-endoconformation. This data indicates the electronic nature ofC2′-substitution has a significant impact on the relative stability ofthese conformations. Substitution of the 2′-O-methyl group increases thepreference for the C3′-endo conformation (when compared toriboguanosine) by about 0.4 kcal/mol at both the HF/6-31G* andMP2/6-31G*//HF16-31G* levels. In contrast, the 2′-S-methyl groupreverses the trend. The C2′-endo conformation is favored by about 2.6kcal/mol at the HF/6-31G* level, while the same difference is reduced to1.41 kcal/mol at the MP2/6-31G*//HF/6-31G* level. For comparison, andalso to evaluate the accuracy of the molecular mechanical force-fieldparameters used for the 2′-O-methyl and 2′-S-methyl substitutednucleosides, we have calculated the gas phase energies of thenucleosides. The results reported in Table 1 indicate that thecalculated relative energies of these nucleosides compare qualitativelywell with the ab initio calculations.

[0108] Additional calculations were also performed to gauge the effectof solvation on the relative stability of nucleoside conformations. Theestimated solvation effect using HF/6-31G* geometries confirms that therelative energetic preference of the four nucleosides in the gas-phaseis maintained in the aqueous phase as well (Table 1). Solvation effectswere also examined using molecular dynamics simulations of thenucleosides in explicit water. From these trajectories, one can observethe predominance of C2′-endo conformation for deoxyriboguanosine and2′-S-methylriboguanosine while riboguanosine and2′-O-methylriboguanosine prefer the C3′-endo conformation. These resultsare in much accord with the available NMR results on2′-S-methylribonucleosides. NMR studies of sugar puckering equilibriumusing vicinal spin-coupling constants have indicated that theconformation of the sugar ring in 2′-S-methylpyrimidine nucleosides showan average of >75% S-character, whereas the corresponding purine analogsexhibit an average of >90% S-pucker [Fraser, A., Wheeler, P., Cook, P.D. and Sanghvi, Y. S., J. HeterocycL. Chem., 1993, 30, 1277-1287]. Itwas observed that the 2′-S-methyl substitution in deoxynucleosideconfers more conformational rigidity to the sugar conformation whencompared with deoxyribonucleosides.

[0109] Structural features of DNA:RNA, OMe_DNA:RNA and SMe_DNA:RNAhybrids were also observed. The average RMS deviation of the DNA:RNAstructure from the starting hybrid coordinates indicate the structure isstabilized over the length of the simulation with an approximate averageRMS deviation of 1.0 Å. This deviation is due, in part, to inherentdifferences in averaged structures (i.e. the starting conformation) andstructures at thermal equilibrium. The changes in sugar puckerconformation for three of the central base pairs of this hybrid are ingood agreement with the observations made in previous NMR studies. Thesugars in the RNA strand maintain very stable geometries in the C3′-endoconformation with ring pucker values near 0°. In contrast, the sugars ofthe DNA strand show significant variability.

[0110] The average RMS deviation of the OMe_DNA:RNA is approximately 1.2Å from the starting A-form conformation; while the SMe_DNA:RNA shows aslightly higher deviation (approximately 1.8 Å) from the starting hybridconformation. The SMe_DNA strand also shows a greater variance in RMSdeviation, suggesting the S-methyl group may induce some structuralfluctuations. The sugar puckers of the RNA complements maintain C3′-endopuckering throughout the simulation. As expected from the nucleosidecalculations, however, significant differences are noted in thepuckering of the OMe_DNA and SMe_DNA strands, with the former adoptingC3′-endo, and the latter, C1′-exo/C2′-endo conformations.

[0111] An analysis of the helicoidal parameters for all three hybridstructures has also been performed to further characterize the duplexconformation. Three of the more important axis-basepair parameters thatdistinguish the different forms of the duplexes, X-displacement,propeller twist, and inclination, are reported in Table 2. Usually, anX-displacement near zero represents a B-form duplex; while a negativedisplacement, which is a direct measure of deviation of the helix fromthe helical axis, makes the structure appear more A-like inconformation. In A-form duplexes, these values typically vary from −4 Åto −5 Å. In comparing these values for all three hybrids, theSMe_DNA:RNA hybrid shows the most deviation from the A-form value, theOMe_DNA:RNA shows the least, and the DNA:RNA is intermediate. A similartrend is also evident when comparing the inclination and propeller twistvalues with ideal A-form parameters. These results are further supportedby an analysis of the backbone and glycosidic torsion angles of thehybrid structures: Glycosidic angles (X) of A-form geometries, forexample, are typically near −159° while B form values are near −102°.These angles are found to be '162°, −133°, and −108° for the OMe_DNA,DNA, and SMe_DNA strands, respectively. All RNA complements adopt an Xangle close to −160°. In addition, “crankshaft” transitions were alsonoted in the backbone torsions of the central UpU steps of the RNAstrand in the SMe_DNA:RNA and DNA;RNA hybrids. Such transitions suggestsome local conformational changes may occur to relieve a less favorableglobal conformation. Taken overall, the results indicate the amount ofA-character decreases as OMe_DNA:RNA>DNA:RNA>SMe_DNA:RNA, with thelatter two adopting more intermediate conformations when compared to A-and B-form geometries. TABLE 2 Average helical parameters derived fromthe last 500 ps of simulation time. (canonical A- and B- form values aregiven for comparison) OMe_(—) SMe_(—) Helicoidal B-DNA B-DNA A-DNA DNA:DNA: DNA: Parameter (x-ray) (fibre) (fibre) RNA RNA RNA X-disp 1.2 0.0−5.3 −4.5 −5.4 −3.5 Inclination −2.3 1.5 20.7 11.6 15.1 0.7 Propeller−16.4 −13.3 −7.5 −12.7 −15.8 −10.3

[0112] Stability of C2′-modified DNA:RNA hybrids was determined.Although the overall stability of the DNA:RNA hybrids depends on severalfactors including sequence-dependencies and the purine content in theDNA or RNA strands DNA:RNA hybrids are usually less stable than RNA:RNAduplexes and, in some cases, even less stable than DNA:DNA duplexes.Available experimental data attributes the relatively lowered stabilityof DNA:RNA hybrids largely to its intermediate conformational naturebetween DNA:DNA (B-family) and RNA:RNA (A-family) duplexes. The overallthermodynamic stability of nucleic acid duplexes may originate fromseveral factors including the conformation of backbone, base-pairing andstacking interactions. While it is difficult to ascertain the individualthermodynamic contributions to the overall stabilization of the duplex,it is reasonable to argue that the major factors that promote increasedstability of hybrid duplexes are better stacking interactions(electrostatic π-π_interactions) and more favorable groove dimensionsfor hydration. The C2′-S-methyl substitution has been shown todestabilize the hybrid duplex. The notable differences in the risevalues among the three hybrids may offer some explanation. While the2′-S-methyl group has a strong influence on decreasing the base-stackingthrough high rise values (˜3.2 Å), the 2′-O-methyl group makes theoverall structure more compact with a rise value that is equal to thatof A-form duplexes (˜2.6 Å). Despite its overall A-like structuralfeatures, the SMe_DNA:RNA hybrid structure possesses an average risevalue of 3.2 Å which is quite close to that of B-family duplexes. Infact, some local base-steps (CG steps) may be observed to have unusuallyhigh rise values (as high as 4.5Å). Thus, the greater destabilization of2′-S-methyl substituted DNA:RNA hybrids may be partly attributed to poorstacking interactions.

[0113] It has been postulated that RNase H binds to the minor groove ofRNA:DNA hybrid complexes, requiring an intermediate minor groove widthbetween ideal A- and B-form geometries to optimize interactions betweenthe sugar phosphate backbone atoms and RNase H. A close inspection ofthe averaged structures for the hybrid duplexes using computersimulations reveals significant variation in the minor groove widthdimensions as shown in Table 3. Whereas the O-methyl substitution leadsto a slight expansion of the minor groove width when compared to thestandard DNA:RNA complex, the S-methyl substitution leads to a generalcontraction (approximately 0.9 Å). These changes are most likely due tothe preferred sugar puckering noted for the antisense strands whichinduce either A- or B-like single strand conformations. In addition tominor groove variations, the results also point to potential differencesin the steric makeup of the minor groove. The O-methyl group points intothe minor groove while the S-methyl is directed away towards the majorgroove. Essentially, the S-methyl group has flipped through the basesinto the major groove as a consequence of C2′-endo puckering. TABLE 3Minor groove widths averaged over the last 500 ps of simulation timeOMe_(—) SMe_(—) Phosphate DNA: DNA: DNA: DNA:RNA RNA:RNA Distance RNARNA RNA (B-form) (A-form) P5-P20 15.27 16.82 13.73 14.19 17.32 P6-P1915.52 16.79 15.73 12.66 17.12 P7-P18 15.19 16.40 14.08 11.10 16.60P8-P17 15.07 16.12 14.00 10.98 16.14 P9-P16 15.29 16.25 14.98 11.6516.93 P10-P15 15.37 16.57 13.92 14.05 17.69

[0114] In addition to the modifications described above, the nucleotidesof the oligonucleotides of the invention can have a variety of othermodification so long as these other modifications do not significantlydetract from the properties described above. Thus, for nucleotides thatare incorporated into oligonucleotides of the invention, thesenucleotides can have sugar portions that correspond tonaturally-occurring sugars or modified sugars. Representative modifiedsugars include carbocyclic or acyclic sugars, sugars having substituentgroups at their 2′ position, sugars having substituent groups at their3′ position, and sugars having substituents in place of one or morehydrogen atoms of the sugar. Other altered base moieties and alteredsugar moieties are disclosed in U.S. Pat. No. 3,687,808 and PCTapplication PCT/US89/02323.

[0115] Altered base moieties or altered sugar moieties also includeother modifications consistent with the spirit of this invention. Sucholigonucleotides are best described as being structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic wild type oligonucleotides. All sucholigonucleotides are comprehended by this invention so long as theyfunction effectively to mimic the structure of a desired RNA or DNAstrand. A class of representative base modifications include tricycliccytosine analog, termed “G clamp” (Lin, et al., J. Am. Chem. Soc. 1998,120, 8531). This analog makes four hydrogen bonds to a complementaryguanine (G) within a helix by simultaneously recognizing theWatson-Crick and Hoogsteen faces of the targeted G. This G clampmodification when incorporated into phosphorothioate oligonucleotides,dramatically enhances antisense potencies in cell culture. Theoligonucleotides of the invention also can includephenoxazine-substituted bases of the type disclosed by Flanagan, et al.,Nat. Biotechnol. 1999, 17(1), 48-52.

[0116] Additional modifications may also be made at other positions onthe oligonucleotide, particularly the 3′position of the sugar on the3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides of theinvention involves chemically linking to the oligonucleotide one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharanet al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBSLett., 1990,259,327;Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0117] As used herein, the term “alkyl” includes but is not limited tostraight chain, branch chain, and cyclic unsaturated hydrocarbon groupsincluding but not limited to methyl, ethyl, and isopropyl groups. Alkylgroups of the present invention may be substituted. Representative alkylsubstituents are disclosed in U.S. Pat. No. 5,212,295, at column 12,lines 41-50, hereby incorporated by reference in its entirety.

[0118] Alkenyl groups according to the invention are to straight chain,branch chain, and cyclic hydrocarbon groups containing at least onecarbon-carbon double bond, and alkynyl groups are to straight chain,branch chain, and cyclic hydrocarbon groups containing at least onecarbon-carbon triply bond. Alkenyl and alkynyl groups of the presentinvention can be substituted.

[0119] Aryl groups are substituted and unsubstituted aromatic cyclicmoieties including but not limited to phenyl, naphthyl, anthracyl,phenanthryl, pyrenyl, and xylyl groups. Alkaryl groups are those inwhich an aryl moiety links an alkyl moiety to a core structure, andaralkyl groups are those in which an alkyl moiety links an aryl moietyto a core structure.

[0120] In general, the term “hetero” denotes an atom other than carbon,preferably but not exclusively N, O, or S. Accordingly, the term“heterocyclic ring” denotes a carbon-based ring system having one ormore heteroatoms (i.e., non-carbon atoms). Preferred heterocyclic ringsinclude, for example but not limited to imidazole, pyrrolidine,1,3-dioxane, piperazine, morpholine rings. As used herein, the term“heterocyclic ring” also denotes a ring system having one or more doublebonds, and one or more heteroatoms. Preferred heterocyclic ringsinclude, for example but not limited to the pyrrolidino ring.

[0121] Oligonucleotides according to the present invention that arehybridizable to a target nucleic acid preferably comprise from about 5to about 50 nucleosides. It is more preferred that such compoundscomprise from about 8 to about 30 nucleosides, with 15 to 25 nucleosidesbeing particularly preferred. As used herein, a target nucleic acid isany nucleic acid that can hybridize with a complementary nucleicacid-like compound. Further in the context of this invention,“hybridization” shall mean hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding between complementarynucleobases. “Complementary” as used herein, refers to the capacity forprecise pairing between two nucleobases. For example, adenine andthymine are complementary nucleobases which pair through the formationof hydrogen bonds. “Complementary” and “specifically hybridizable,” asused herein, refer to precise pairing or sequence complementaritybetween a first and a second nucleic acid-like oligomers containingnucleoside subunits. For example, if a nucleobase at a certain positionof the first nucleic acid is capable of hydrogen bonding with anucleobase at the same position of the second nucleic acid, then thefirst nucleic acid and the second nucleic acid are considered to becomplementary to each other at that position. The first and secondnucleic acids are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleobaseswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule. It is understood that an oligomeric compound of the inventionneed not be 100% complementary to its target RNA sequence to bespecifically hybridizable. An oligomeric compound is specificallyhybridizable when binding of the oligomeric compound to the target RNAmolecule interferes with the normal function of the target RNA to causea loss of utility, and there is a sufficient degree of complementarityto avoid non-specific binding of the oligomeric compound to non-targetsequences under conditions in which specific binding is desired, i.e.under physiological conditions in the case of in vivo assays ortherapeutic treatment, or in the case of in vitro assays, underconditions in which the assays are performed.

[0122] The oligonucleotides of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism should be contacted with an oligonucleotidehaving a sequence that is capable of specifically hybridizing with astrand of nucleic acid coding for the undesirable protein. Treatments ofthis type can be practiced on a variety of organisms ranging fromunicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular control is susceptible to therapeutic and/orprophylactic treatment in accordance with the invention. Seeminglydiverse organisms such as bacteria, yeast, protozoa, algae, all plantsand all higher animal forms, including warm-blooded animals, can betreated. Further, each cell of multicellular eukaryotes can be treated,as they include both DNA-RNA transcription and RNA-protein translationas integral parts of their cellular activity. Furthermore, many of theorganelles (e.g. mitochondria and chloroplasts) of eukaryotic cells alsoinclude transcription and translation mechanisms. Thus, single cells,cellular populations or organelles can also be included within thedefinition of organisms that can be treated with therapeutic ordiagnostic oligonucleotides.

[0123] Some representative therapeutic indications and other uses forthe compounds of the invention are as follows:

[0124] One therapeutic indication of particular interest is psoriasis.Psoriasis is a common chronic and recurrent disease characterized bydry, well-circumscribed, silvery, scaling papules and plaques of varioussizes. The disease varies in severity from a few lesions to widespreaddermatosis with disabling arthritis or exfoliation. The ultimate causeof psoriasis is not known, but the thick scaling that occurs is probablydue to increased epidermal cell proliferation (The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 2283-2285, Berkow et al., eds.,Rahway, N.J., 1987). Inhibitors of Protein Kinase C (PKC) have beenshown to have both antiproliferative and anti-inflammatory effects invitro. Some antipsoriasis drugs, such as cyclosporin A and anthralin,have been shown to inhibit PKC, and inhibition of PKC has been suggestedas a therapeutic approach to the treatment of psoriasis (Hegemann, L.and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., pp.357-368,CRC Press, Boca Raton, Fla., 1992). Antisense compounds targeted toProtein Kinase C (PKC) proteins are described in U.S. Pat. Nos.5,620,963 to Cook et al. and 5,681,747 to Boggs et al.

[0125] Another type of therapeutic indication of interest isinflammatory disorders of the skin. These occur in a variety of formsincluding, for example, lichen planus, toxic epidermal necrolyis (TEN),ertythema multiforme and the like (The Merck Manual of Diagnosis andTherapy, 15th Ed., pp. 2286-2292, Berkow et al., eds., Rahway, N.J.,1987). Expression of ICAM-1 has been associated with a variety ofinflammatory skin disorders such as allergic contact dermatitis, fixeddrug eruption, lichen planus and psoriasis (Ho et al., J. Am. Acad.Dermatol., 1990, 22, 64; Griffiths et al., Am. J. Pathology, 1989, 135,1045; Lisby et al., Br. J Dermatol., 1989, 120, 479; Shiohara et al.,Arch. Dermatol., 1989, 125, 1371; Regezi et al., Oral Surg. Oral Med.Oral Pathol., 1996, 81, 682). Moreover, intraperitoneal administrationof a monoclonal antibody to ICAM-1 decreases ovalbumin-inducedeosinophil infiltration into skin in mice (Hakugawa et al., J.Dermatol., 1997, 24,73). Antisense compounds targeted to ICAM-1 aredescribed in U.S. Pat. Nos. 5,514,788 and 5,591,623, and co-pending U.S.patent applications Ser. Nos. 09/009,490 and 09/062,416, Jan. 20, 1998and Apr. 17, 1998, respectively, all to Bennett et al.

[0126] Other antisense targets for skin inflammatory disorders areVCAM-1 and PECAM-1. Intraperitoneal administration of a monoclonalantibody to VCAM-1 decreases ovalbumin-induced eosinophil infiltrationinto the skin of mice (Hakugawa et al., J. Dermatol., 1997, 24, 73).Antisense compounds targeted to VCAM-1 are described in U.S. Pat. Nos.5,514,788 and 5,591,623. PECAM-1 proteins are glycoproteins which areexpressed on the surfaces of a variety of cell types (for reviews, seeNewman, J. Clin. Invest., 1997, 99, 3 and DeLisser et al., Immunol.Today, 1994, 15, 490). In addition to directly participating incell-cell interactions, PECAM-1 apparently also regulates the activityand/or expression of other molecules involved in cellular interactions(Litwin et al., J. Cell Biol., 1997, 139, 219) and is thus a keymediator of several cell:cell interactions. Antisense compounds targetedto PECAM-1 are described in co-pending U.S. patent application Ser. No.09/044,506, filed Mar. 19, 1998, by Bennett et al.

[0127] Another type of therapeutic indication of interest foroligonucleotides encompasses a variety of cancers of the skin.Representative skin cancers include benign tumors (warts, moles and thelike) and malignant tumors such as, for example, basal cell carcinoma,squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi'ssarcoma and the like (The Merci Manual of Diagnosis and Therapy, 15thEd., pp. 2301-2310, Berkow et al., eds., Rahway, N.J., 1987). A numberof molecular targets involved in tumorigenesis, maintenance of thehyperproliferative state and metastasis are targeted to prevent orinhibit skin cancers, or to prevent their spread to other tissues.

[0128] The ras oncogenes are guanine-binding proteins that have beenimplicated in cancer by, e.g., the fact that activated ras oncogeneshave been found in about 30% of human tumors generally; this figureapproached 100% in carcinomas of the exocrine pancreas (for a review,see Downward, Trends in Biol. Sci., 1990, 15, 469). Antisense compoundstargeted to H-ras and K-ras are described in U.S. Pat. No. 5,582,972 toLima et al., 5,582,986 to Monia et al. and 5,661,134 to Cook et al., andin published PCT application WO 94/08003.

[0129] Protein Kinase C (PKC) proteins have also been implicated intumorigenesis. Antisense compounds targeted to Protein Kinase C (PKC)proteins are described in U.S. Pat. Nos. 5,620,963 to Cook et al. and5,681,747 to Boggs et al. Also of interest are AP-1 subunits and JNKproteins, particularly in regard to their roles in tumorigenesis andmetastasis. The process of metastasis involves a sequence of eventswherein (1) a cancer cell detaches from its extracellular matrices, (2)the detached cancer cell migrates to another portion of an animal'sbody, often via the circulatory system, and (3) attaches to a distal andinappropriate extracellular matrix, thereby created a focus from which asecondary tumor can arise. Normal cells do not possess the ability toinvade or metastasize and/or undergo apoptosis (programmed cell death)if such events occur (Ruoslahti, Sci. Amer., 1996, 275, 72). However,many human tumors have elevated levels of activity of one or more matrixmetalloproteinases (MMPs) (Stetler-Stevenson et al., Annu. Rev. CellBiol., 1993, 9, 541; Bernhard et al., Proc. Natl. Acad. Sci. (U.S.A.),1994,91, 4293. The MMPs are a family of enzymes which have the abilityto degrade components of the extracellular matrix (Birkedal-Hansen,Current Op. Biol., 1995, 7, 728). In particular, one member of thisfamily, matrix metalloproteinase-9 (MMP-9), is often found to beexpressed only in tumors and other diseased tissues (Himelstein et al.,Invasion & Metastasis, 1994, 14, 246).

[0130] Several studies have shown that regulation of the MMP-9 gene maybe controlled by the AP-1 transcription factor (Kerr et al., Science,1988, 242, 1242; Kerr et al., Cell, 1990, 61, 267; Gum et al.,J. Biol.Chem., 1996, 271, 10672; Hua et al., Cancer Res., 1996, 56, 5279).Inhibition of AP-1 function has been shown to attenuate MMP-9 expression(U.S. patent application Ser. No. 08/837,201). AP-1 is a heterodimericprotein having two subunits, the gene products of fos and jun. Antisensecompounds targeted to c-fos and c-jun are described in co-pending U.S.patent application Ser. No. 08/837,201, filed Mar. 14, 1997, by Dean etal.

[0131] Furthermore, AP-1 is itself activated in certain circumstances byphosphorylation of the Jun subunit at an amino-terminal position by JunN-terminal kinases (JNKs). Thus, inhibition of one or more JNKs isexpected to result in decreased AP-1 activity and, consequentially,reduced MMP expression. Antisense compounds targeted to JNKs aredescribed in co-pending U.S. patent application Ser. No. 08/910,629,filed Aug. 13, 1997, by Dean et al.

[0132] Infectious diseases of the skin are caused by viral, bacterial orfungal agents. In the case of Lyme disease, the tick borne causativeagent thereof, the spirochete Borrelia burgdorferi, up-regulates theexpression of ICAM-1, VCAM-1 and ELAM-1 on endothelial cells in vitro(Boggemeyer et al., Cell Adhes. Comm., 1994, 2, 145). Furthermore, ithas been proposed that the mediation of the disease by theanti-inflammatory agent prednisolone is due in part to mediation of thisup-regulation of adhesion molecules (Hurtenbach et al., Int. J.Immunopharmac., 1996, 18, 281). Thus, potential targets for therapeuticmediation (or prevention) of Lyme disease include ICAM-1, VCAM-1 andELAM-1 (supra).

[0133] Other infectious disease of the skin which are tractable totreatment using the compositions and methods of the invention includedisorders resulting from infection by bacterial, viral or fungal agents(The Merck Manual of Diagnosis and Therapy, 15th Ed., pp. 2263-2277,Berkow et al., eds., Rahway, N.J., 1987). With regards to infections ofthe skin caused by fungal agents, U.S. Pat. No. 5,691,461 providesantisense compounds for inhibiting the growth of Candida albicans.

[0134] With regards to infections of the skin caused by viral agents,U.S. Pat. Nos. 5,166,195, 5,523,389 and 5,591,600 provideoligonucleotide inhibitors of Human Immunodeficiency Virus (HIV). U.S.Pat. No. 5,004,810 provides oligomers capable of hybridizing to herpessimplex virus Vmw65 mRNA and inhibiting its replication. U.S. Pat. No.5,194,428 and 5,580,767 provide antisense compounds having antiviralactivity against influenza virus. U.S. Pat. No. 4,806,463 providesantisense compounds and methods using them to inhibit HTLV-IIIreplication. U.S. Pat. Nos. 4,689,320, 5,442,049, 5,591,720 and5,607,923 are directed to antisense compounds as antiviral agentsspecific to cytomegalovirus (CMV). U.S. Pat. No. 5,242,906 providesantisense compounds useful in the treatment of latent Epstein-Barr virus(EBV) infections. U.S. Pat. Nos. 5,248,670, 5,514,577 and 5,658,891provide antisense compounds useful in the treatment of herpes virusinfections. U.S. Pat. Nos. 5,457,189 and 5,681,944 provide antisensecompounds useful in the treatment of papilloma virus infections. Theantisense compounds disclosed in these patents, which are hereinincorporated by reference, may be used with the compositions of theinvention to effect prophylactic, palliative or therapeutic relief fromdiseases caused or exacerbated by the indicated pathogenic agents.

[0135] Antisense oligonucleotides employed in the compositions of thepresent invention may also be used to determine the nature, function andpotential relationship of various genetic components of the body todisease or body states in animals. Heretofore, the function of a genehas been chiefly examined by the construction of loss-of-functionmutations in the gene (i. e., “knock-out” mutations) in an animal (e.g.,a transgenic mouse). Such tasks are difficult, time-consuming and cannotbe accomplished for genes essential to animal development since the“knock-out” mutation would produce a lethal phenotype. Moreover, theloss-of-function phenotype cannot be transiently introduced during aparticular part of the animal's life cycle or disease state; the“knock-out” mutation is always present. “Antisense knockouts,” that is,the selective modulation of expression of a gene by antisenseoligonucleotides, rather than by direct genetic manipulation, overcomesthese limitations (see, for example, Albert et al., Trends inPharmacological Sciences, 1994, 15, 250). In addition, some genesproduce a variety of mRNA transcripts as a result of processes such asalternative splicing; a “knock-out” mutation typically removes all formsof mRNA transcripts produced from such genes and thus cannot be used toexamine the biological role of a particular mRNA transcript. Antisenseoligonucleotides have been systemically administered to rats in order tostudy the role of the N-methyl-D-aspartate receptor in neuronal death,to mice in order to investigate the biological role of protein kinaseC-a, and to rats in order to examine the role of the neuropeptide Y1receptor in anxiety (Wahlestedt et al., Nature, 1993, 363:260; Dean etal., Proc. Natl. Acad. Sci. U.S.A., 1994, 91:11762; and Wahlestedt etal., Science, 1993, 259:528, respectively). In instances where complexfamilies of related proteins are being investigated, “antisenseknockouts” (i.e., inhibition of a gene by systemic administration ofantisense oligonucleotides) may represent the most accurate means forexamining a specific member of the family (see, generally, Albert etal., Trends Pharmacol Sci., 1994, 15:250). By providing compositions andmethods for the simple non-parenteral delivery of oligonucleotides andother nucleic acids, the present invention overcomes these and othershortcomings.

[0136] The administration of therapeutic or pharmaceutical compositionscomprising the oligonucleotides of the invention is believed to bewithin the skill of those in the art. In general, a patient in need oftherapy or prophylaxis is administered a composition comprising acompound of the invention, commonly in a pharmaceutically acceptablecarrier, in doses ranging from 0.01 ug to 100 g per kg of body weightdepending on the age of the patient and the severity of the disorder ordisease state being treated. Dosing is dependent on severity andresponsiveness of the disease state to be treated, with the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution or prevention of the disease state isachieved. Optimal dosing schedules can be calculated from measurementsof drug accumulation in the body of the patient. Persons of ordinaryskill can easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual antisense compounds, and can generally beestimated based on EC₅₀s found to be effective in in vitro and in vivoanimal models.

[0137] In the context of the invention, the term “treatment regimen” ismeant to encompass therapeutic, palliative and prophylactic modalitiesof administration of one or more compositions of the invention. Aparticular treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease or disorder,its severity and the overall condition of the patient, and may extendfrom once daily to once every 20 years. Following treatment, the patientis monitored for changes in his/her condition and for alleviation of thesymptoms of the disorder or disease state. The dosage of the compositionmay either be increased in the event the patient does not respondsignificantly to current dosage levels, or the dose may be decreased ifan alleviation of the symptoms of the disorder or disease state isobserved, or if the disorder or disease state has been ablated.

[0138] An optimal dosing schedule is used to deliver a therapeuticallyeffective amount of the oligonucleotide of the invention. The term“therapeutically effective amount,” for the purposes of the invention,refers to the amount of oligonucleotide-containing pharmaceuticalcomposition which is effective to achieve an intended purpose withoutundesirable side effects (such as toxicity, irritation or allergicresponse). Although individual needs may vary, determination of optimalranges for effective amounts of pharmaceutical compositions is withinthe skill of the art. Human doses can be extrapolated from animalstudies (Katocs et al., Chapter 27 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990). Generally, the dosage required to provide an effective amount ofa pharmaceutical composition, which can be adjusted by one skilled inthe art, will vary depending on the age, health, physical condition,weight, type and extent of the disease or disorder of the recipient,frequency of treatment, the nature of concurrent therapy (if any) andthe nature and scope of the desired effect(s) (Nies et al., Chapter 3In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).

[0139] Following successful treatment, it may be desirable to have thepatient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the bioactive agent is administered inmaintenance doses, ranging from 0.01 ug to 100 g per kg of body weight,once or more daily, to once every 20 years. For example, in the case ofin individual known or suspected of being prone to an autoimmune orinflammatory condition, prophylactic effects may be achieved byadministration of preventative doses, ranging from 0.01 ug to 100 g perkg of body weight, once or more daily, to once every 20 years. In likefashion, an individual may be made less susceptible to an inflammatorycondition that is expected to occur as a result of some medicaltreatment, e.g., graft versus host disease resulting from thetransplantation of cells, tissue or an organ into the individual.

[0140] Prophylactic modalities for high risk individuals are alsoencompassed by the invention. As used herein, the term “high riskindividual” is meant to refer to an individual for whom it has beendetermined, via, e.g., individual or family history or genetic testing,that there is a significantly higher than normal probability of beingsusceptible to the onset or recurrence of a disease or disorder. Forexample, a subject animal could have a personal and/or family medicalhistory that includes frequent occurrences of a particular disease ordisorder. As another example, a subject animal could have had such asusceptibility determined by genetic screening according to techniquesknown in the art (see, e.g., U.S. Congress, Office of TechnologyAssessment, Chapter 5 In: Genetic Monitoring and Screening in theWorkplace, OTA-BA-455, U.S. Government Printing Office, Washington,D.C., 1990, pages 75-99). As part of a treatment regimen for a high riskindividual, the individual can be prophylactically treated to preventthe onset or recurrence of the disease or disorder. The term“prophylactically effective amount” is meant to refer to an amount of apharmaceutical composition which produces an effect observed as theprevention of the onset or recurrence of a disease or disorder.Prophylactically effective amounts of a pharmaceutical composition aretypically determined by the effect they have compared to the effectobserved when a second pharmaceutical composition lacking the activeagent is administered to a similarly situated individual.

[0141] For therapeutic use the oligonucleotide analog is administered toan animal suffering from a disease modulated by some protein. It ispreferred to administer to patients suspected of suffering from such adisease an amount of oligonucleotide analog that is effective to reducethe symptomology of that disease. One skilled in the art can determineoptimum dosages and treatment schedules for such treatment regimens.

[0142] For use in diseases modulated by protein that portion of DNA orRNA which codes for the protein whose formation or activity is to bemodulated is targeted. The targeting portion of the composition to beemployed is, thus, selected to be complementary to the preselectedportion of DNA or RNA, that is to be an antisense oligonucleotide forthat portion.

[0143] It is generally preferred to administer the therapeutic agents inaccordance with this invention internally such as orally, intravenously,or intramuscularly. Other forms of administration, such astransdermally, topically, or intralesionally may also be useful.Inclusion in suppositories may also be useful. Use of pharmacologicallyacceptable carriers is also preferred for some embodiments.

[0144] This invention is also directed to methods for the selectivebinding of RNA for research and diagnostic purposes wherein it is usefulto effect strand cleavage utilizing enzymatic RNase H cleavage whileconcurrently effecting modulation of binding affinity and or nucleaseresistance. Such selective is accomplished by interacting such RNA orDNA with compositions of the invention which are resistant todegradative nucleases and which hybridize more strongly and with greaterfidelity than known oligonucleotides or oligonucleotide analogs.

[0145] Oligonucleotides according to the invention can be assembled insolution or through solid-phase reactions, for example, on a suitableDNA synthesizer utilizing nucleosides, phosphoramidites and derivatizedcontrolled pore glass (CPG) according to the invention and/or standardnucleotide precursors. In addition to nucleosides that include a novelmodification of the inventions other nucleoside within anoligonucleotide may be further modified with other modifications at the2′ position. Precursor nucleoside and nucleotide precursors used to formsuch additional modification may carry substituents either at the 2′ or3′ positions. Such precursors may be synthesized according to thepresent invention by reacting appropriately protected nucleosidesbearing at least one free 2′ or 3′ hydroxyl group with an appropriatealkylating agent such as, but not limited to, alkoxyalkyl halides,alkoxylalkylsulfonates, hydroxyalkyl halides, hydroxyalkyl sulfonates,aminoalkyl halides, aminoalkyl sulfonates, phthalimidoalkyl halides,phthalimidoalkyl sulfonates, alkylaminoalkyl halides, alkylaminoalkylsulfonates, dialkylaminoalkyl halides, dialkylaminoalkylsulfonates,dialkylaminooxyalkyl halides, dialkylaminooxyalkyl sulfonates andsuitably protected versions of the same. Preferred halides used foralkylating reactions include chloride, bromide, fluoride and iodide.Preferred sulfonate leaving groups used for alkylating reactionsinclude, but are not limited to, benzenesulfonate, methylsulfonate,tosylate, p-bromobenzenesulfonate, triflate, trifluoroethylsulfonate,and (2,4-dinitroanilino)benzenesulfonate.

[0146] Suitably protected nucleosides can be assembled intooligonucleotides according to known techniques. See, for example,Beaucage et al., Tetrahedron, 1992, 48, 2223.

[0147] The ability of oligonucleotides to bind to their complementarytarget strands is compared by determining the melting temperature(T_(m)) of the hybridization complex of the oligonucleotide and itscomplementary strand. The melting temperature (T_(m)), a characteristicphysical property of double helices, denotes the temperature (in degreescentigrade) at which 50% helical (hybridized) versus coil (unhybridized)forms are present. T_(m) is measured by using the UV spectrum todetermine the formation and breakdown (melting) of the hybridizationcomplex. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands. The structure-stability relationships of a large number ofnucleic acid modifications have been reviewed (Freier and Altmann, Nucl.Acids Research, 1997, 25, 4429-443).

[0148] The relative binding ability of the oligonucleotides of thepresent invention was determined using protocols as described in theliterature (Freier and Altmann, Nucl. Acids Research, 1997, 25,4429-443). Typically absorbance versus temperature curves weredetermined using samples containing 4 uM oligonucleotide in 100 mM Na+,10 mM phosphate, 0.1 mM EDTA, and 4 uM complementary, length matchedRNA.

[0149] The in vivo stability of oligonucleotides is an important factorto consider in the development of oligonucleotide therapeutics.Resistance of oligonucleotides to degradation by nucleases,phosphodiesterases and other enzymes is therefore determined. Typical invivo assessment of stability of the oligonucleotides of the presentinvention is performed by administering a single dose of 5 mg/kg ofoligonucleotide in phosphate buffered saline to BALB/c mice. Bloodcollected at specific time intervals post-administration is analyzed byHPLC or capillary gel electrophoresis (CGE) to determine the amount ofoligonucleotide remaining intact in circulation and the nature the ofthe degradation products.

[0150] Heterocyclic bases amenable to the present invention include bothnaturally and non-naturally occurring nucleobases and heterocycles. Arepresentative list includes adenine, guanine, cytosine, uridine, andthymine, as well as other synthetic and natural nucleobases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo,oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adeninesand guanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine. Further heterocyclic bases include thosedisclosed in U.S. Pat. No. 3,687,808, those disclosed in the ConciseEncyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed byEnglisch, et al., Angewandte Chemie, International Edition 1991, 30,613.

[0151] Additional objects, advantages, and novel features of thisinvention will become apparent to those skilled in the art uponexamination of the following examples, which are not intended to belimiting. All oligonucleotide sequences are listed in a standard 5′ to3′ order from left to right.

EXAMPLE 1 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyl uridine and5′-O-DMT-3′-O-(2-methoxyethyl)5-methyl uridine

[0152] 2′,3′-O-dibutylstannylene 5-methyl uridine (345 g) (prepared asper: Wagner et al., J. Org. Chem., 1974, 39, 24) was alkylated with2-methoxyethyl bromide (196 g) in the presence of tetrabutylammoniumiodide (235 g) in DMF (3 L) at 70° C. to give a mixture of 2′-O— and3′-O-(2-methoxyethyl)-5-methyl uridine (150 g) in nearly 1:1 ratio ofisomers. The mixture was treated with DMT chloride (110 g, DMT-Cl) inpyridine (1 L) to give a mixture of the 5′-O-DMT-nucleosides. After thestandard work-up the isomers were separated by silica gel columnchromatography. The 2′-isomer eluted first, followed by the 3′-isomer.

EXAMPLE 25′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl-uridine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0153]5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyluridine (5 g, 0.008 mol)was dissolved in CH₂Cl₂ (30 mL) and to this solution, under argon,diisopropylaminotetrazolide (0.415 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (3.9 mL) were added. The reaction was stirred overnight.The solvent was evaporated and the residue was applied to silica columnand eluted with ethyl acetate to give 3.75 g title compound.

EXAMPLE 3 5′-O-DMT-3′-O-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine

[0154] 5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl uridine (15 g) wastreated with 150 mL anhydrous pyridine and 4.5 mL of acetic anhydrideunder argon and stirred overnight. Pyridine was evaporated and theresidue was partitioned between 200 mL of saturated NaHCO₃ solution and200 mL of ethylacetate. The organic layer was dried (anhydrous MgSO₄)and evaporated to give 16 g of2′-acetoxy-5′-O-(DMT)-3′-O-(2-methoxyethyl)-5-methyl uridine.

[0155] To an ice-cold solution of triazole (19.9 g) in triethylamine (50mL) and acetonitrile (150 mL), with mechanical stirring, 9 mL of POCl₃was added dropwise. After the addition, the ice bath was removed and themixture stirred for 30 min. The2′-acetoxy-5′-O-(DMT)-3′-O-(2-methoxyethyl) -5-methyl uridine (16 g in50 mL CH₃CN) was added dropwise to the above solution with the receivingflask kept at ice bath temperatures. After 2 hrs, TLC indicated a fastermoving nucleoside, C-4-triazole-derivative. The reaction flask wasevaporated and the nucleoside was partitioned between ethylacetate (500mL) and NaHCO₃ (500 mL). The organic layer was washed with saturatedNaCl solution, dried (anhydrous NgSO₄) and evaporated to give 15 g ofC-4-triazole nucleoside. This compound was then dissolved in 2:1 mixtureof NH₄OH/dioxane (100 mL:200 mL) and stirred overnight. TLC indicateddisappearance of the starting material. The solution was evaporated anddissolved in methanol to crystallize out 9.6 g of5′-O-(DMT)-3′-O-(2-methoxyethyl) 5-methyl cytidine.

[0156] 5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl cytidine (9.6 g, 0.015mol) was dissolved in 50 mL of DMF and treated with 7.37 g of benzoicanhydride. After 24 hrs of stirring, DMF was evaporated and the residuewas loaded on silica column and eluted with 1:1 hexane:ethylacetate togive the desired nucleoside.

EXAMPLE 45′-O-DMT-3′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0157]5′-O-DMT-3′-O-(2-methoxyethyl)-N-benzoyl-5-methyl-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditewas obtained from the above nucleoside using the phosphitylationprotocol described for the corresponding 5-methyl-uridine derivative.

EXAMPLE 5 N⁶-Benzoyl-5′-O-(DMT)-3′-O-(2-methoxyethyl) adenosine

[0158] A solution of adenosine (42.74 g, 0.16 mol) in dry dimethylformamide (800 ML) at 5° C. was treated with sodium hydride (8.24 g, 60%in oil prewashed thrice with hexanes, 0.21 mol). After stirring for 30min, 2-methoxyethyl bromide (0.16 mol) was added over 20 min. Thereaction was stirred at 5° C. for 8 h, then filtered through Celite. Thefiltrate was concentrated under reduced pressure followed bycoevaporation with toluene (2×10 mL). The residue was adsorbed on silicagel (100 g) and chromatographed (800 g, chloroform-methanol 9:14:1).Selected fractions were concentrated under reduced pressure and theresidue was a mixture of 2′-O-(2-(methoxyethyl) adenosine and3′-O-(2-methoxyethyl) adenosine in the ratio of 4:1.

[0159] The above mixture (0.056 mol) in pyridine (100 mL) was evaporatedunder reduced pressure to dryness. The residue was redissolved inpyridine (560 mL) and cooled in an ice water bath. Trimethylsilylchloride (36.4 mL, 0.291 mol) was added and the reaction was stirred at5° C. for 30 min. Benzoyl chloride (33.6 mL, 0.291 mol) was added andthe reaction was allowed to warm to 25° C. for 2 h and then cooled to 5°C. The reaction was diluted with cold water (112 mL) and after stirringfor 15 min, concentrated ammonium hydroxide (112 Ml) was added. After 30min, the reaction was concentrated under reduced pressure (below 30° C.)followed by coevaporation with toluene (2×100 mL). The residue wasdissolved in ethyl acetate-methanol (400 mL, 9:1) and the undesiredsilyl by-products were removed by filtration. The filtrate wasconcentrated under reduced pressure and then chromatographed on silicagel (800 g, chloroform-methanol 9:1). Selected fractions were combined,concentrated under reduced pressure and dried at 25° C./0.2 mmHg for 2 hto give pure N⁶-Benzoyl-2′-O-(2-methoxyethyl) adenosine and pureN⁶Benzoyl-3′-O-(2-methoxyethyl) adenosine.

[0160] A solution of N⁶-Benzoyl-3′-O-(2-methoxyethyl) adenosine (11.0 g,0.285 mol) in pyridine (100 mL) was evaporated under reduced pressure toan oil. The residue was redissolved in dry pyridine (300 mL) and DMT-Cl(10.9 g, 95%, 0.31 mol) was added. The mixture was stirred at 25° C. for16 h and then poured onto a solution of sodium bicarbonate (20 g) in icewater (500 mL). The product was extracted with ethyl acetate (2×150 mL).The organic layer was washed with brine (50 mL), dried over sodiumsulfate (powdered) and evaporated under reduced pressure (below 40 C.).The residue was chromatographed on silica gel (400 g, ethylacetate-acetonitrile-triethylamine 99:1:195:5:1). Selected fractionswere combined, concentrated under reduced pressure and dried at 25°C./0.2 mmHg to give 16.8 g (73%) of the title compound as a foam. TheTLC was homogenous.

EXAMPLE 6 [N⁶-Benzoyl-5′-O(DM3′-O-(2-methoxyethyl)adenosin-2′-O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite

[0161] The title compound was prepared in the same manner as the5-methyl-cytidine and 5-methyluridine analogs of Examples 2 and 4 bystarting with the title compound of Example 5. Purification using ethylacetate-hexanes-triethylamine 59:40:1 as the chromatography eluent gave67% yield of the title compound as a solid foam. The TLC was homogenous.³¹P-NMR (CDCl₃, H₃PO₄ std.) δ147.89; 148.36 (diastereomers).

EXAMPLE 7 5′O-(DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl) guanosine

[0162] A. 2.6-Diaminopurine riboside

[0163] To a 2 L stainless steel Parr bomb was added guanosine hydrate(100 g, 0.35 mol, Aldrich), hexamethyl) disilazane (320 mL, 1.52 mol,4.4 eq.), trimethyl) silyl trifiouromethanesulfonate (8.2 mL), andtoluene (350 mL). The bomb was sealed and partially submerged in an oilbath (170° C.; internal T 150° C., 150 psi) for 5 days. The bomb wascooled in a dry ice/acetone bath and opened. The contents weretransferred with methanol (300 mL) to a flask and the solvent wasevaporated under reduced pressure. Aqueous methanol (50%, 1.2 L)wasadded. The resulting brown suspension was heated to reflux for 5 h. Thesuspension was concentrated under reduced pressure to one half volume inorder to remove most of the methanol. Water (600 mL) was added and thesolution was heated to reflux, treated with charcoal (5 g) and hotfiltered through Celite. The solution was allowed to cool to 25° C. Theresulting precipitate was collected, washed with water (200 mL) anddried at 90° C./0.2 mmHg for 5 h to give a constant weight of 87.4 g(89%) of tan, crystalline solid; mp 247° C. (shrinks), 255° C. (dec,lit. (1) mp 250-252° C.); TLC homogenous (Rf 0.50, isopropanol-ammoniumhydroxide-water 16:3:1 ); PMR (DMSO), δ5.73 (d, 2, 2-NH₂), 5.78 (s, 1,H-1), 6.83 (br s, 2, 6-NH₂).

[0164] B. 21-O-(2-methoxyethyl)-2,6-diaminopurine riboside and3′-O-(2-methoxyethyl)-2,6-diaminopurine riboside

[0165] To a solution of 2,6-diaminopurine riboside (10.0 g, 0.035 mol)in dry dimethyl formamide (350 mL) at 0° C. under an argon atmospherewas added sodium hydride (60% in oil, 1.6 g, 0.04 mol). After 30 min.,2-methoxyethyl bromide (0.44 mol) was added in one portion and thereaction was stirred at 25° C. for 16 h. Methanol (10 mL) was added andthe mixture was concentrated under reduced pressure to an oil (20 g).The crude product, containing a ratio of 4:1 of the 2′/3′ isomers, waschromatographed on silica gel (500 g, chloroform-methanol 4:1). Theappropriate fractions were combined and concentrated under reducedpressure to a semi-solid (12 g). This was triturated with methanol (50mL) to give a white, hygroscopic solid. The solid was dried at 40°C./0.2 mmHg for 6 h to give a pure 2′ product and the pure 3′ isomer,which were confirmed by NMR.

[0166] C. 3′-O-2-(methoxyethyl)guanosine

[0167] With rapid stirring, 3′-O-(2-methoxyethyl)-2,6-diaminopurineriboside (0.078 mol) was dissolved in monobasic sodium phosphate buffer(0.1 M, 525 mL, pH 7.3-7.4) at 25° C. Adenosine deaminase (Sigma typeII, 1 unit/mg, 350 mg) was added and the reaction was stirred at 25° C.for 60 h. The mixture was cooled to 5° C. and filtered. The solid waswashed with water (2×25 mL) and dried at 60° C./0.2 mmHg for 5 h to give10.7 g of first crop material. A second crop was obtained byconcentrating the mother liquors under reduced pressure to 125 mL,cooling to 5° C., collecting the solid, washing with cold water (2×20mL) and drying as above to give 6.7 g of additional material for a totalof 15.4 g (31% from guanosine hydrate) of light tan solid; TLC purity97%.

[0168] D. N²-Isobutyryl-3′-O-2-(methoxyethyl)guanosine

[0169] To a solution of3′-O-2-(methoxyethyl)guanosine(18.1 g, 0.0613mol) in pyridine (300 mL) was added trimethyl silyl chloride (50.4 mL,0.46 mol). The reaction was stirred at 25° C. for-16 h. Isobutyrylchloride (33.2 mL, 0.316 mol) was added and the reaction was stirred for4 h at 25° C. The reaction was diluted with water (25 mL). Afterstirring for 30 min, ammonium hydroxide (concentrated, 45 mL) was addeduntil pH 6 was reached. The mixture was stirred in a water bath for 30min and then evaporated under reduced pressure to an oil. The oil wassuspended in a mixture of ethyl acetate (600 mL) and water (100 mL)until a solution formed. The solution was allowed to stand for 17 h at25° C. The resulting precipitate was collected, washed with ethylacetate (2×50 mL) and dried at 60° C./0.2 mmHg for 5 h to give 16.1 g(85%) of tan solid; TLC purity 98%.

[0170] E. 5′-O-DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl) guanosine

[0171] A solution of N²-Isobutyryl-3′-O-2-(methoxyethyl) guanosine(0.051 mol) in pyridine (150 mL) was evaporated under reduced pressureto dryness. The residue was redissolved in pyridine (300 mL) and cooledto 10-15° C. DMT-Cl (27.2 g, 95%, 0.080 mol) was added and the reactionwas stirred at 25° C. for 16 h. The reaction was evaporated underreduced pressure to an oil, dissolved in a minimum of methylene chlorideand applied on a silica gel column (500 g). The product was eluted witha gradient of methylene chloride-triethylamine (99:1) to methylenechloride-methanol-triethylamine (99:1:1). Selected fractions werecombined, concentrated under reduced pressure and dried at 40° C./0.2mmHg for 2 h to afford 15 g (15.5% from guanosine hydrate) of tan foam;TLC purity 98%.

EXAMPLE 8 [5′-O-(DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl)guanosin-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0172] The protected nucleoside from Example 7 (0.0486 mol) was placedin a dry 1 L round bottom flask containing a Teflon stir-bar. The flaskwas purged with argon. Anhydrous methylene chloride (400 mL) wascannulated into the flask to dissolve the nucleoside. Previously vacuumdried N,N-diisopropylaminohydrotetrazolide (3.0 g, 0.0174 mol) was addedunder argon. With stirring,bis-N,N-diisopropyl-aminocyanoethylphosphoramidite (18.8 g, 0.0689 mol)was added via syringe over 1 min (no exotherm noted). The reaction wasstirred under argon at 25° C. for 16 h. After verifying the completionof the reaction by TLC, the reaction was transferred to a separatoryfunnel (1 L). The reaction flask was rinsed with methylene chloride(2×50 mL). The combined organic layer was washed with saturated aq.sodium bicarbonate (200 mL) and then brine (200 mL). The organic layerwas dried over sodium sulfate (50 g, powdered) for 2 h. The solution wasfiltered and concentrated under reduced pressure to a viscous oil. Theresulting phosphoramidite was purified by silica gel flashchromatography (800 g, ethyl acetate-triethylamine 99:1). Selected werecombined, concentrated under reduced pressure, and dried at 25 C./0.2mmHg for 16 h to give 18.0 g (46%, 3% from guanosine hydrate) of solidfoam TLC homogenous. ³¹P-NMR (CDCl₃, H₃PO₄ std.) δ147.96; 148.20(diastereomers).

EXAMPLE 9 5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl-uridine-2′-O-succinate

[0173] 5′-O-DMT-3′-O-(2-methoxyethyl)-thymidine was first succinylatedon the 2′-position. Thymidine nucleoside (4 mmol) was reacted with 10.2mL dichloroethane, 615 mg (6.14 mmol) succinic anhydride, 570 μL (4.09mmol) triethylamine, and 251 mg (2.05 mmol) 4-dimethylaminopyridine. Thereactants were vortexed until dissolved and placed in heating block at55° C. for approximately 30 minutes. Completeness of reaction checked bythin layer chromatography (TLC). The reaction mixture was washed threetimes with cold 10% citric acid followed by three washes with water. Theorganic phase was removed and dried under sodium sulfate. Succinylatednucleoside was dried under P₂O₅ overnight in vacuum oven.

EXAMPLE 10 5′-O-DMT-3′-methoyethyl-5methyl-uridine-2-O-succinoyl LinkedLCA CPG5′-O-DMT-3′

[0174] O-(2-methoxyethyl)-2′-O-succinyl-thymidine was coupled tocontrolled pore glass (CPG). 1.09 g (1.52 mmol) of the succinate weredried overnight in a vacuum oven along with 4-dimethylaminopyridine(DMAP), 2,2′-dithiobis (5-nitro-pyridine) (dTNP), triphenylphosphine(TPP), and pre-acid washed CPG (controlled pore glass). After about 24hours, DMAP (1.52 mmol, 186 mg) and acetonitrile (13.7 mL) were added tothe succinate. The mixture was stirred under an atmosphere of argonusing a magnetic stirrer. In a separate flask, dTNP (1.52 mmol, 472 mg)was dissolved in acetonitrile (9.6 mL) and dichloromethane (4.1 mL)under argon. This reaction mixture was then added to the succinate. Inanother separate flask, TPP (1.52 mmol, 399 mg) was dissolved inacetonitrile (37 mL) under argon. This mixture was then added to thesuccinate/DMAP/dTNP reaction mixture. Finally, 12.23 g pre-acid washedLCA CPG (loading=115.2 μmol/g) was added to the main reaction mixture,vortexed shortly and placed on shaker for approximately 3 hours. Themixture was removed from the shaker after 3 hours and the loading waschecked. A small sample of CPG was washed with copious amounts ofacetonitrile, dichloromethane, and then with ether. The initial loadingwas found to be 63 μmol/g (3.9 mg of CPG was cleaved withtrichloroacetic acid, the absorption of released trityl cation was readat 503 nm on a spectrophotometer to determine the loading.) The wholeCPG sample was then washed as described above and dried under P₂O₅overnight in vacuum oven. The following day, the CPG was capped with 25mL CAP A (tetrahydrofuran/acetic anhydride) and 25 mL CAP B(tetrahydrofuran/pyridine/1methyl imidazole) for approximately 3 hourson shaker. Filtered and washed with dichloromethane and ether. The CPGwas dried under P₂O₅ overnight in vacuum oven. After drying, 12.25 g ofCPG was isolated with a final loading of 90 μmol/g.

EXAMPLE 11 3′-O-Methoxyethyl-5-methyl-N-benzoyl-cytidine-2′-O-succinate

[0175] 5′-O-DMT-3′-O-(2-methoxy)ethyl-N-benzoyl-cytidine was firstsuccinylated on the 2′-position. Cytidine nucleoside (4 mmol) wasreacted with 10.2 mL dichloroethane, 615 mg (6.14 mmol) succinicanhydride, 570 μL (4.09 mmol) triethylamine, and 251 mg (2.05 mmol)4-dimethylaminopyridine. The reactants were vortexed until dissolved andplaced in a heating block at 55° C. for approximately 30 minutes.Completeness of reaction was checked by thin layer chromatography (TLC).The reaction mixture was washed three times with cold 10% citric acidfollowed by three washes with water. The organic phase was removed anddried under sodium sulfate. The succinylated nucleoside was dried underP₂O₅ overnight in vacuum oven.

EXAMPLE 125′-O-DMT-3-O-methoxyethyl-5-methyl-N-benzoyl-cytidine-2′-O-succinoyllinked LCA CPG

[0176] 5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N⁴-benzoyl cytidinewas coupled to controlled pore glass (CPG). 1.05 g (1.30 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMA), 2,2′-dithiobis (5-nitro-pyridine) (dTNP),triphenylphosphine (TPP), and pre-acid washed CPG (controlled poreglass). The following day, DMAP (1.30 mmol, 159 mg) and acetonitrile(11.7 mL) were added to the succinate. The mixture was “mixed” by amagnetic stirrer under argon. In a separate flask, dTNP (1.30 mmol, 400mg) was dissolved in acetonitrile (8.2 mL) and dichloromethane (3.5 mL)under argon. This reaction mixture was then added to the succinate. Inanother separate flask, TPP (1.30 mmol, 338 mg) was dissolved inacetonitrile (11.7 mL) under argon. This mixture was then added to thesuccinate/DMAP/dTNP reaction mixture. Finally, 10.46 g pre-acid washedLCA CPG (loading=115.2 μmol/g) were added to the main reaction mixture,vortexed shortly and placed on shaker for approximately 2 hours. Aportion was removed from shaker after 2 hours and the loading waschecked. A small sample of CPG was washed with copious amounts ofacetonitrile, dichloromethane, and then with ether. The initial loadingwas found to be 46 μmol/g. (3.4 mg of CPG were cleaved withtrichloroacetic acid). The absorption of released trityl cation was readat 503 nm on a spectrophotometer to determine the loading. The whole CPGsample was then washed as described above and dried under P₂O₅ overnightin vacuum oven. The following day, the CPG was capped with 25 mL CAP. A(tetrahydrofuran/acetic anhydride) and 25 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 3 hourson a shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 10.77 g of CPG was isolated with a final loading of 63 μmol/g.

EXAMPLE 13 5′-O-DMT-3′-O-methoxyethyl-N6benzoyl-adenosine-2′-O-succinate

[0177] 5′-O-DMT-3′-O-2-methoxyethyl)-N⁶-benzoyl adenosine was firstsuccinylated on the 2′-position. 3.0 g (4.09 mmol) of the adenosinenucleoside were reacted with 10.2 mL dichloroethane, 615 mg (6.14 mmol)succinic anhydride, 570 μL (4.09 mmol) triethylamine, and 251 mg (2.05mmol) 4-dimethylaminopyridine. The reactants were vortexed untildissolved and placed in heating block at 55° C. for approximately 30minutes. Completeness of reaction was checked by thin layerchromatography (TLC). The reaction mixture was washed three times withcold 10% citric acid followed by three washes with water. The organicphase was removed and dried under sodium sulfate. Succinylatednucleoside was dried under P₂O₅ overnight in vacuum oven.

EXAMPLE 145′-O-DMT-3′-O-(2-methoxyethyl)-N6benzoyl-adenosine-2′-O-succinoyl LinkedLCA CPG

[0178] Following succinylation,5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N⁶-benzoyl adenosine wascoupled to controlled pore glass (CPG). 3.41 g (4.10 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMAP), 2,2′-dithiobis (5-nitro-pyridine)(dTNP), triphenylphosphine (TPP), and pre-acid washed CPG (controlledpore glass). The following day, DMAP (4.10 mmol, 501 mg) andacetonitrile (37 mL) were added to the succinate. The mixture was“mixed” by a magnetic stirrer under argon. In a separate flask, dTNP(4.10 mmol, 1.27 g) was dissolved in acetonitrile (26 mL) anddichloromethane (11 mL) under argon. This reaction mixture was thenadded to the succinate. In another separate flask, TPP (4.10 mmol, 1.08g) was dissolved in acetonitrile (37 mL) under argon. This mixture wasthen added to the succinate/DMAP/dTNP reaction mixture. Finally, 33 gpre-acid washed LCA CPG (loading=115.2 μmol/g) were added to the mainreaction mixture, vortexed shortly and placed on shaker forapproximately 20 hours. Removed from shaker after 20 hours and theloading was checked. A small sample of CPG was washed with copiousamounts of acetonitrile, dichloromethane, and then with ether. Theinitial loading was found to be 49 μmol/g. (2.9 mg of CPG were cleavedwith trichloroacetic acid). The absorption of released trityl cation wasread at 503 nm on a spectrophotometer to determine the loading. Thewhole CPG sample was then washed as described above and dried under P₂O₅overnight in vacuum oven. The following day, the CPG was capped with 50mL CAP A (tetrahydrofuran/acetic anhydride) and 50 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 1 houron the shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 33.00 g of CPG was obtained with a final loading of 66 μmol/g.

EXAMPLE 155′-O-DMT-3-O-(2-methoxyethyl)-N2-isobutyryl-guanosine-2′-O-succinate

[0179] 5′-O-DMT-3′-O-(2-methoxyethyl)1-N²-isobutyryl guanosine wassuccinylated on the 2′-sugar position. 3.0 g (4.20mmol)of the guanosinenucleoside were reacted with 10.5 mL dichloro-ethane, 631 mg (6.30 mmol)succinic anhydride, 585 μL (4.20 mmol) triethylamine, and 257 mg (2.10mmol) 4-dimethylaminopyridine. The reactants were vortexed untildissolved and placed in heating block at 55° C. for approximately 30minutes. Completeness of reaction checked by thin layer chromatography(TLC). The reaction mixture was washed three times with cold 10% citricacid followed by three washes with water. The organic phase was removedand dried under sodium sulfate. The succinylated nucleoside was driedunder P₂O₅ overnight in vacuum oven.

EXAMPLE 16

[0180] 5′-O-DMT-3′-O-methoxyethyl-N2-isobutyryl-guanosine2′-O-succinoylLinked LCA CPG

[0181] Following succinylation,5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N2-benzoyl guanosine wascoupled to controlled pore glass (CPG). 3.42 g (4.20 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMAP), 2,2′-dithiobis (5-nitro-pyridine)(dTNP), triphenylphosphine (TPP), and pre-acid washed CPG (controlledpore glass). The following day, DMAP (4.20 mmol, 513 mg) andacetonitrile (37.5 mL) were added to the succinate. The mixture was“mixed” by a magnetic stirrer under argon. In a separate flask, dTNP(4.20 mmol, 1.43 g) was dissolved in acetonitrile (26 mL) anddichloromethane (11 mL) under argon. This reaction mixture was thenadded to the succinate. In another separate flask, TPP (4.20 mmol, 1.10g) was dissolved in acetonitrile (37.5 mL) under argon. This mixture wasthen added to the succinate/DMAP/dTNP reaction mixture. Finally, 33.75 gpre-acid washed LCA CPG (loading=115.2 μmol/g) were added to the mainreaction mixture, vortexed shortly and placed on shaker forapproximately 20 hours. Removed from shaker after 20 hours and theloading was checked. A small sample of CPG was washed with copiousamounts of acetonitrile, dichloromethane, and then with ether. Theinitial loading was found to be 64 μmol/g. (3.4 mg of CPG were cleavedwith trichloroacetic acid). The absorption of released trityl cation wasread at 503 nm on a spectrophotometer to determine the loading. The CPGwas then washed as described above and dried under P₂O₅ overnight invacuum oven. The following day, the CPG was capped with 50 mL CAP A(tetrahydrofuran/acetic anhydride) and 50 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 1 houron a shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 33.75 g. of CPG was isolated with a final loading of 72 μmol/g.

EXAMPLE 17 5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine

[0182] 2′,3′-O-Dibutyl stannylene-uridine was synthesized according tothe procedure of Wagner et. al., J. Org. Chem., 1974, 39,24. Thiscompound was dried over P₂O₅ under vacuum for 12 hours. To a solution ofthis compound (29 g, 42.1 mmol) in 200 mL of anhydrous DMF were added(16.8 g, 55 mmol) of 6-bromohexyl phthalimide and 4.5 g of sodium iodideand the mixture was heated at 130° C. for 16 hours under argon. Thereaction mixture was evaporated, co-evaporated once with toluene and thegummy tar residue was applied on a silica column (500 g). The column waswashed with 2 L of EtOAc followed by eluting with 10% methanol(MeOH):90% EtOAc. The product, 2′-and 3′-isomers ofO-hexyl-6-N-phthalimido uridine, eluted as an inseparable mixture(R_(f)=0.64 in 10% MeOH in EtOAc). By ¹³C NMR, the isomeric ration wasabout 55% of the 2′ isomer and about 45% of the 3′ isomer. The combinedyield was 9.2 g (46.2%). This mixture was dried under vacuum andre-evaporated twice with pyridine. It was dissolved in 150 mL anhydrouspyridine and treated with 7.5 g of DMT-Cl (22.13 mmol) and 500 mg ofdimethylaminopyridine (DMAP). After 2 hours, thin layer chromatography(TLC; 6:4 EtOAc:Hexane) indicated complete disappearance of the startingmaterial and a good separation between 2′ and 3′ isomers (R_(f)=0.29 forthe 2′ isomer and 0.12 for the 3′ isomer). The reaction mixture wasquenched by the addition of 5 mL of CH₃OH and evaporated under reducedpressure. The residue was dissolved in 300 mL CH₂Cl₂, washedsuccessively with saturated NaHCO₃ followed by saturated NaCl solution.It was dried over Mg₂SO₄ and evaporated to give 15 g of a brown foamwhich was purified on a silica gel (500 g) to give 6.5 g of the2′-isomer and 3.5 g of the 3′ isomer.

EXAMPLE 185′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine-2′-O-(2-cyanoethyl-N,N,-diisopropyl)phosphoramidite

[0183] 5′-DMT-3′-O-[hexyl-(6-phthalimido)]uridine (2 g, 2.6 mmol) wasdissolved in 20 mL anhydrous CH₂Cl₂. To this solutiondiisopropylaminotetrazolide (0.2 g, 1.16 mmol) and 2.0 mL2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphoramidite (6.3 mmol) wereadded with stirred overnight. TLC (1:1 EtOAc/hexane) showed completedisappearance of starting material. The reaction mixture was transferredwith CH₂C1 ₂ and washed with saturated NaHCO₃ (100 mL), followed bysaturated NaCl solution. The organic layer was dried over anhydrousNa₂SO₄ and evaporated to yield 3.8 g of a crude product, which waspurified in a silica column (200 g) using 1:1 hexane/EtOAc to give 1.9 g(1.95 mmol, 74% yield) of the desired phosphoramidite.

EXAMPLE 19 Preparation of5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine2′-O-succinoyl-aminopropylCPG

[0184] Succinylated and capped aminopropyl controlled pore glass (CPG;500 Å pore diameter, aminopropyl CPG, 1.0 grams prepared according toDamha et. al., Nucl Acids Res. 1990, 18, 3813.) was added to 12 mLanhydrous pyridine in a 100 mL round-bottom flask.1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide (DEC; 0.38 grams, 2.0mmol)], triethylamine (TEA; 100 μl, distilled over CaH₂),dimethylaminopyridine (DMAP; 0.012 grams, 0.1 mmol) and nucleoside5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]uridine (0.6 grams, 0.77 mmol) wereadded under argon and the mixture shaken mechanically for 2 hours.Additional nucleoside (0.20 grams) was added and the mixture shaken for24 hours. The CPG was filtered off and washed successively withdichloromethane, triethylamine, and dichloromethane. The CPG was thendried under vacuum, suspended in 10 mL piperidine and shaken 15 minutes.The CPG was filtered off, washed thoroughly with dichloromethane andagain dried under vacuum. The extent of loading (determined byspectrophotometric assay of DMT cation in 0.3 M p-toluenesulfonic acidat 498 nm) was approximately 28 μmol/g. The5′-O-(DMT)-3′-O-[hexyl-(6-phthalimido]uridine-2′-O-succinoyl-aminopropyl controlled pore glass was used tosynthesize the oligomers in an ABI 380B DNA synthesizer usingphosphoramidite chemistry standard conditions. A four base oligomer5′-GACU*-3′ was used to confirm the structure of 3′-O-hexylamine tetherintroduced into the oligonucleotide by NMR. As expected a multipletsignal was observed between 1.0-1.8 ppm in ¹H NMR.

EXAMPLE 20 5′-O-DMT-3′-O-[hexylamino]-uridine

[0185] 5′-O-(DMT)-3′-O-[hexyl-(6-phthalimido)] uridine (4.5 grams, 5.8mmol) is dissolved in 200 mL methanol in a 500 mL flask. Hydrazine (1ml, 31 mmol) is added to the stirring reaction mixture. The mixture isheated to 60-65° C. in an oil bath and refluxed 14 hours. The solvent isevaporated in vacuo and the residue is dissolved in dichloromethane (250mL) and extracted twice with an equal volume NH₄OH. The organic layer isevaporated to yield the crude product which NMR indicates is notcompletely pure. R_(f)=0 in 100% ethyl acetate. The product is used insubsequent reactions without further purification.

EXAMPLE 21 3′-O-[Propyl-(3-phthalimido)]-adenosine

[0186] To a solution of adenosine (20.0 g, 75 mmol) in drydimethylformamide (550 ml) at 5° C. was added sodium hydride (60% oil,4.5 g, 112 mmol). After one hour, N-(3-bromopropyl)-phthalimide (23.6 g,86 mmol) was added and the temperature was raised to 30° C. and held for16 hours. Ice is added and the solution evaporated in vacuo to a gum.The gum was partitioned between water and ethyl acetate (4×300 mL). Theorganic phase was separated, dried, and evaporated in vacuo and theresultant gum chromatographed on silica gel (95/5 CH₂Cl₂/MeOH) to give awhite solid (5.7 g) of the 2′-O-(propylphthalimide)adenosine. Theefractions containing the 3′-O-(propylphthalimide)adenosine werechromatographed a second time on silica gel using the same solventsystem.

[0187] Crystallization of the 2′-O-(propylphthalimide)adenosinefractions from methanol gave a crystalline solid, m.p. 123-124C. ¹H NMR(400 MHZ: DMSO-d₆)δ1.70(m, 2H, CH₂), 3.4-3.7 (m, 6, C₅, OCH₂, Phth CH₂),3.95 (q, 1H, C₄H), 4.30 (q, 1H, C₅H), 4.46 (t, 1H, C₂H), 5.15 (d, 1H,C_(3′)OH), 5.41 (t, 1H, C_(5′)OH), 5.95 (d, 1H, C_(1′)H) 7.35 (s, 2HNH₂), 7.8 (brs, 4H, Ar), 8.08 (s, 1H, C₂H) and 8.37 (s, 1H, C₈H). Anal.Calcd. C₂₁H₂₂N₆O₆: C, 55.03; H, 4.88; N, 18.49. Found: C, 55.38; N,18.46.

[0188] Crystallization of the 3′-O-(propylphthalimide)adenosinefractions from H₂O afforded an analytical sample, m.p. 178-179C. ¹H NMR(400 MHZ: DMSO-d₆) δ5.86 (d, 1H, H-1′).

EXAMPLE 22 3′-O-[Propyl-(3-phthalimido)]-N6-benzoyl-adenosine

[0189] 3′-O-(3-propylphthalimide)adenosine is treated with benzoylchloride in a manner similar to the procedure of Gaffiey, et al.,Tetrahedron Lett. 1982, 23, 2257. Purification of crude material bychromatography on silica gel (ethyl acetate-methanol) gives the titlecompound.

EXAMPLE 23 3′-O-[Propyl-(3-phthalimido)]-5′-O-DMT-N6-benzoyl-adenosine

[0190] To a solution of 3′-O-(propyl-3-phthalimide)-N⁶-benzoyladenosine(4.0 g) in pyridine (250 ml) is added DMT-Cl (3.3 g). The reaction isstirred for 16 hours. The reaction is added to ice/water/ethyl acetate,the organic layer separated, dried, and concentrated in vacuo and theresultant gum chromatographed on silica gel (ethyl acetate-methanoltriethylamine) to give the title compound.

EXAMPLE 243′-O-[Propyl-(3-phthalimido)]-51-O-DMT-N6-Benzoyl-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0191] 3′-O-(Propyl-3-phthalimide)-5′-O-DMT-N⁶-benzoyladenosine istreated with (β-cyanoethoxy)chloro-N, N-diisopropyl)aminophosphane in amanner similar to the procedure of Seela, et al., Biochemistry 1987, 26,2233. Chromatography on silica gel (EtOAc/hexane) gives the titlecompound as a white foam.

EXAMPLE 25 3′-O-(Aminopropyl)-adenosine

[0192] A solution of 3′-O-(propyl-3-phthalimide)adenosine (8.8 g, 19mmol), 95% ethanol (400 mL) and hydrazine (10 mL, 32 mmol) is stirredfor 16 hrs at room temperature. The reaction mixture is filtered andfiltrate concentrated in vacuo. Water (150 mL) is added and acidifiedwith acetic acid to pH 5.0. The aqueous solution is extracted with EtOAc(2×30 mL) and the aqueous phase is concentrated in vacuo to afford thetitle compound as a HOAc salt.

EXAMPLE 26

[0193] 3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

[0194] A solution of 3′-O-(propylamino)adenosine in methanol (50 mL) andtriethylamine (15 mL, 108 mmol) is treated with ethyl trifluoroacetate(18 mL, 151 mmol). The reaction is stirred for 16 hrs and thenconcentrated in vacuo and the resultant gum chromatographed on silicagel (9/1, EtOAc/MeOH) to give the title compound.

EXAMPLE 27 N6-Dibenzoyl-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

[0195] 3′-O-[3-(N-trifluoroacetamido)propyl]adenosine is treated as perExample 22 using a Jones modification wherein tetrabutylammoniumfluoride is utilized in place of ammonia hydroxide in the work up. Thecrude product is purified using silica gel chromatography (EtOAc/MeOH1/1) to give the title compound.

EXAMPLE 28N6-Dibenzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

[0196] DMT-Cl (3.6 g, 10.0 mmol) is added to a solution ofN⁶-(dibenzoyl)-3′-O-[3-(N-trifluoro-acetamido)propyl)adenosine inpyridine (100 mL) at room temperature and stirred for 16 hrs. Thesolution is concentrated in vacuo and chromatographed on silica gel(EtOAc/TEA 99/1) to give the title compound.

EXAMPLE 29 N6-Dibenzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0197] A solution ofN⁶-(dibenzoyl)-5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosinein dry CH₂Cl₂ is treated with bis-N,N-diisopropylamino cyanoethylphosphite (1.1 eqiv) and N,N-diisopropylaminotetrazolide (catalyticamount) at room temperature for 16 hrs. The reaction is concentrated invacuo and chromatographed on silica gel (EtOAc/hexane/TEA 6/4/1) to givethe title compound.

EXAMPLE 30 3′-O-(butylphthalimido)-adenosine

[0198] The title compound is prepared as per Example 21, usingN-(4-bromobutyl)phthalimide in place of the 1-bromopropane.Chromatography on silica gel (EtOAC-MeOH) gives the title compound. ¹HNMR (200 MHZ, DMSO-d₆) 67 5.88 (d, 1H, C₁H).

EXAMPLE 31 N6-Benzoyl-3′-O-(butylphthalimido)-adenosine

[0199] Benzoylation of 3′-O-(butylphthalimide)adenosine as per Example22 gives the title compound.

EXAMPLE 32 N6-Benzoyl-5′-O-DMT-3′-O-(butylphthalimido)-adenosine

[0200] The title compound is prepared from3′-O-(butyl-phthalimide)-N⁶-benzoyladenosine as per Example 22.

EXAMPLE 33N6-Benzoyl-5′-O-DMT-3′-O-(butylphthalimido)-Adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0201] The title compound is prepared from3′-O-(butylphthalimide)-5′-O-DMT-N⁶-benzoyladenosine as per Example 24.

EXAMPLE 34 3′-O-(Pentylphthalimido)-adenosine

[0202] The title compound is prepared as per Example 21, usingN-(5-bromopentyl)phthalimide. The crude material from the extraction ischromatographed on silica gel using CHCl₃/MeOH (95/5) to give a mixtureof the 2′ and 3′ isomers. The 2′ isomer is recrystallized from EtOH/MeOH8/2. The mother liquor is rechromatographed on silica gel to afford the3′ isomer. 2′-O-(Pentylphthalimido)adenosine: M.P. 159-160° C. Anal.Calcd. for C₂₃H₂₄N₆O₅: C, 57.26; H, 5.43; N, 17.42. Found: C, 57.03; H,5.46; N, 17.33. 3′-O-(Pentylphthalimido)adenosine: ¹H NMR (DMSO-d₆) 675.87 (d, 1H, H-1′).

EXAMPLE 35 N6-Benzoyl-3′-O-(pentylphthalimido)-adenosine

[0203] Benzoylation of 3′-O-(pentylphthalimido)adenosine is achieved asper the procedure of Example 22 to give the title compound.

EXAMPLE 36 N6Benzoyl-5′-O-DMT-3′-O-(pentylphthalimido)-adenosine

[0204] The title compound is prepared from3′-O-(pentyl-phthalimide)-N⁶-benzoyladenosine as per the procedure ofExample 23. Chromatography on silica gel (ethylacetate, hexane,triethylamine), gives the title compound.

EXAMPLE 37N6-Benzoyl-5′-O-DMT-31-O-(pentylphthalimido)-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0205] The title compound is prepared from3′-O-(pentyl-phthalimide)-5′-O-(DMT)-N⁶-benzoyladenosine as per theprocedure of Example 24 to give the title compound.

EXAMPLE 38 3′-O-(Propylphthalimido)uridine

[0206] A solution of uridine-tin complex (48.2 g, 115 mmol) in dry DM(150 ml) and N-(3-bromopropyl)phthalimide (46 g, 172 mmol) was heated at130° C. for 6 hrs. The crude product was chromatographed directly onsilica gel CHCl₃/MeOH 95/5. The isomer ratio of the purified mixture was2′/3′ 81/19. The 2′ isomer was recovered by crystallization from MeOH.The filtrate was rechromatographed on silica gel using CHCl₃CHCl₃/MeOH(95/5) gave the 3′ isomer as a foam. 2′-O-(Propylphthalimide)uridine:Analytical sample recrystallized from MeOH, m.p. 165.5-166.5C., ¹H NMR(200 MHZ, DMSO-d₆) 67 1.87 (m, 2H, CH₂), 3.49-3.65 (m, 4H, C_(2′)H),3.80-3.90 (m, 2H, C_(3′),H₁C_(4′)H), 4.09(m, 1H, C_(2′),H), 5.07 (d, 1h,C_(3′)OH), 5.16 (m, 1H, C_(5′)OH), 5.64 (d, 1H, CH=), 7.84 (d, 1H,C_(1′)H), 7.92 (bs, 4H, Ar), 7.95 (d, 1H, CH=) and 11.33 (s, 1H, ArNH).Anal. C₂₀H₂₁N₃H₈, Calcd. C, 55.69; H, 4.91;, N, 9.74. Found, C, 55.75;H, 5.12; N, 10.01. 3′-O-(Propylphthalimide)uridine: ¹H NMR (DMSO-d₆)δ5.74 (d, 1H, H-1′).

EXAMPLE 39 3′-O-(Aminopropyl)-uridine

[0207] The title compound is prepared as per the procedure of Example25.

EXAMPLE 40

[0208] 3′-O-[3-(N-trifluoroacetamido)propyl]-uridine

[0209] 3′-O-(Propylamino)uridine is treated as per the procedure ofExample 26 to give the title compound.

EXAMPLE 41 5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-uridine

[0210] 3′-O-[3-(N-trifluoroacetamido)propyl]uridine is treated as perthe procedure of Example 28 to give the title compound.

EXAMPLE 425′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-uridine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0211] 5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]uridine istreated as per the procedure of Example 29 to give the title compound.

EXAMPLE 43 3′-O-(Propylphthalimido)-cytidine

[0212] The title compounds were prepared as per the procedure of Example21. 2′-O-(propylphthalimide)cytidine: ¹H NMR (200 MHZ, DMSO-d₆) δ5.82(d, 1H, C_(1′)H). 3′-O-(propylphthalimide)cytidine: IH NMR (200 MHZ,DMSO-d₆) δ5.72 (d, 1H, C_(1′)H).

EXAMPLE 44 3′-O-(Aminopropyl)cytidine

[0213] 3′-O-(Propylphthalimide)cytidine is treated as per the procedureof Example 25 to give the title compound.

EXAMPLE 45 3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

[0214] 3′-O-(Propylamino)cytidine is treated as per the procedure ofExample 26 to give the title compound.

EXAMPLE 46 N4-Benzoyl-3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

[0215] 3′-O-[3-(N-trifluoroacetamido)propyl]cytidine is treated as perthe procedure of Example 27 to give the title compound.

EXAMPLE 47N4-Benzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

[0216] N⁴-(Benzoyl)-3′-O-[3-(N-trifluoroacetamido)propyl]cytidine istreated as per the procedure of Example 28 to give the title compound.

EXAMPLE 48N4-Benzoyl-5′-O-DMT-3′-O-[3-(N-trifuoroacetamido)propyl]-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0217]N⁴-(Benzoyl)-5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]cytidine istreated as per the procedure of Example 29 to give the title compound.

EXAMPLE 49 General Procedures for Oligonucleotide Synthesis

[0218] Oligonucleotides were synthesized on a Perseptive BiosystemsExpedite 8901 Nucleic Acid Synthesis System. Multiple 1-μmol syntheseswere performed for each oligonucleotide. Trityl groups were removed withtrichloroacetic acid (975 μL over one minute) followed by anacetonitrile wash. All standard amidites (0.1 M) were coupled twice percycle (total coupling time was approximately 4 minutes). All novelamidites were dissolved in dry acetonitrile (100 mg of amidite/1 mLacetonitrile) to give approximately 0.08-0.1 M solutions. Total couplingtime was approximately 6 minutes (105 μL of amidite delivered).1-H-tetrazole in acetonitrile was used as the activating agent. Excessamidite was washed away with acetonitrile. (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO, 1.0 g CSO/8.72 mL dry acetonitrile) was used tooxidize (4 minute wait step)phosphodiester linkages while3H-1,2-benzodithiole-3-one-1,1-dioxide (Beaucage reagent, 3.4 g Beaucagereagent/200 mL acetonitrile) was used to oxidize (one minute wait step)phosphorothioate linkages. Unreacted functionalities were capped with a50:50 mixture of tetrahydrofuran/acetic anhydride andtetrahydrofuran/pyridine/1-methyl imidazole. Trityl yields were followedby the trityl monitor during the duration of the synthesis. The finalDMT group was left intact. The oligonucleotides were deprotected in 1 mL28.0-30% ammonium hydroxide (NH₄OH) for approximately 16 hours at 55° C.Oligonucleotides were also made on a larger scale (20 μmol/synthesis).Trityl groups were removed with just over 8 mL of trichloroacetic acid.All standard amidites (0.1 M) were coupled twice per cycle (13 minutecoupling step). All novel amidites were also coupled four times percycle but the coupling time was increased to approximately 20 minutes(delivering 480 μL of amidite). Oxidation times remained the same butthe delivery of oxidizing agent increased to approximately 1.88 mL percycle. Oligonucleotides were cleaved and deprotected in 5 mL 28.0-30%NH₄0H at 55° C, for approximately 16 hours. TABLE I3′-O-(2-methoxyethyl) containing 2′-5′ linked o- ligonucleotides. SEQ IDNO. (ISIS) Back- Chem- # # Sequence (5′ 3′)¹ bone istry 4 (17176)ATG-CAT-TCT-GCC-CCC-AAG- P = S 3′-O-MOE GA* 5 (17177)ATG-CAT-TCT-GCC-CCC-AAG- P = S 3′-O-MOE G*A* 6 (17178)ATG-CAT-TCT-GCC-CCC- P = S/ 3′-O-MOE AAG_(O)-G* _(O) A* P = O 7 (17179)A*TG-CAT-TCT-GCC-CCC- P = S 3′-O-MOE AAG-GA* 8 (17180)A*TG-CAT-TCT-GCC-CCC- P = S 3′-O-MOE AAG-G*A* 9 (17181) A*_(O)TG-CAT-TCT-GCC-AAA- P = S/ 3′-O-MOE AAG_(O)-G* _(O) A* P = O 10(21415) A*T*G-CAT-TCT-GCC-AAA- P = S 3′-O-MOE AAG-G*A* 11 (21416) A*_(O) T* _(O)G-CAT-TCT-GCC-AAA- P = S/ 3′-O-MOE AAG_(O)-G* _(O) A* P = O(21945) A*A*A* P = O 3′-O-MOE (21663) A*A*A*A* P = O 3′-O-MOE (20389)A*U*C*G* P = O 3′-O-MOE 12 (20390) C*G*C*-G*A*A*-T*T*C*-G* P = O3′-O-MOE C*G*

EXAMPLE 50 General Procedure for Purification of Oligonucleotides

[0219] Following cleavage and deprotection step, the crudeoligonucleotides (such as those synthesized in Example 49) were filteredfrom CPG using Gelman 0.45 μm nylon acrodisc syringe filters. ExcessNH₄OH was evaporated away in a Savant AS160 automatic speed vac. Thecrude yield was measured on a Hewlett Packard 8452A Diode ArraySpectrophotometer at 260 nm. Crude samples were then analyzed by massspectrometry (MS) on a Hewlett Packard electrospray mass spectrometerand by capillary gel electrophoresis (CGE) on a Beckmann P/ACE system5000. Trityl-on oligonucleotides were purified by reverse phasepreparative high performance liquid chromatography (HPLC). HPLCconditions were as follows: Waters 600 E 991 detector; Waters Delta PakC4 column (7.8×300 mm); Solvent A: 50 mM triethylammonium acetate(TEA-Ac), pH 7.0; B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient:5% B for first five minutes with linear increase in B to 60% during thenext 55 minutes. Larger oligo yields from the larger 20 μmol syntheseswere purified on larger HPLC columns (Waters Bondapak HC18HA) and theflow rate was increased to 5.0 mL/min. Appropriate fractions werecollected and solvent was dried down in speed vac. Oligonucleotides weredetritylated in 80% acetic acid for approximately 45 minutes andlyophilized again. Free trityl and excess salt were removed by passingdetritylated oligonucleotides through Sephadex G-25 (size exclusionchromatography) and collecting appropriate samples through a Pharmaciafraction collector. Solvent again evaporated away in speed vac. Purifiedoligonucleotides were then analyzed for purity by CGE, HPLC (flow rate:1.5 mL/min; Waters Delta Pak C4 column, 3.9×300mm), and MS. The finalyield was determined by spectrophotometer at 260 nm. TABLE II Physicalcharacteristics of 3'-O-(2-methoxyethyl) containing 2'-5' linkedoligonucleotides. HPLC² Expected Observed T_(R) Purified Mass Mass(min.) #Ods(260 nm) 17176 6440.743 6440.300 23.47 3006 17177 6514.8146513.910 23.67 3330 17178 6482.814 6480.900 23.06 390 17179 6513.7986513.560 23.20 3240 17180 6588.879 6588.200 23.96 3222 17181 6540.8796539.930 23.01 21415 6662.976 6662.700 24.18 4008 21416 6598.9696597.800 23.01 3060 21945 1099.924 1099.300 19.92 121 21663 1487.3241486.800 20.16 71 20389 1483.000 1482.000 62 20390 4588.000 4591.000 151#Solvent A: 50 mM TEA-Ac, pH 7.0; B: 100% acetonitrile; 1.5 mL/min. flowrate; Gradient: #5% B for first five minutes with linear increase in Bto 60% during the next 55 minutes.

EXAMPLE 51 T_(m) Studies on Modified Oligonucleotides

[0220] Oligonucleotides synthesized in Examples 49 and 50 were evaluatedfor their relative ability to bind to their complementary nucleic acidsby measurement of their melting temperature (T_(m)). The meltingtemperature (T_(m)), a characteristic physical property of doublehelices, denotes the temperature (in degrees centigrade) at which 50%helical (hybridized) versus coil (unhybridized) forms are present. T_(m)is measured by using the UV spectrum to determine the formation andbreakdown (melting) of the hybridization complex. Base stacking, whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

[0221] Selected test oligonucleotides and their complementary nucleicacids were incubated at a standard concentration of 4 μM for eacholigonucleotide in buffer (100 mM NaCl, 10 mM sodium phosphate, pH 7.0,0.1 mM EDTA). Samples were heated to 90 ° C. and the initial absorbancetaken using a Guilford Response II Spectrophotometer (Coming). Sampleswere then slowly cooled to 15° C. and then the change in absorbance at260 nm was monitored with heating during the heat denaturationprocedure. The temperature was increased by 1 degree ° C./absorbancereading and the denaturation profile analyzed by taking the 1^(st)derivative of the melting curve. Data was also analyzed using atwo-state linear regression analysis to determine the Tm=s. The resultsof these tests for the some of the oligonucleotides from Examples 49 and50 are shown in Table III below. TABLE III Tm Analysis ofOligonucleotides SEQ # ID: Back- Mods # NO. (ISIS) bone Link- 2′- # #Sequence (5′-3′) T_(m)8 ages 5′ 13 (11061) ATG-CAT-TCT-GCC- P = S 61.4 00 CCC-AAG-GA 4 (17176) ATG-CAT-TCT-GCC- P = S 61.4 1 0 CCC-AAG-GA* 5(17177) ATG-CAT-TCT-GCC- P = S 61.3 2 1 CCC-AAG-G*A* 6 (17178)ATG-CAT-TCT-GCC- P = S/ 61.8 2 1 CCC-AAG_(O)-G* _(O) A* P = O 7 (17179)A*TG-CAT-TCT-GCC- P = S 61.1 2 1 CCC-AAG-GA* 8 (17180) A*TG-CAT-TCT-GCC-P = S 61.0 3 2 CCC-AAG-G*A* 9 (17181) A* _(O)TG-CAT-TCT-GCC- P = S/ 61.83 2 AAA-AAG_(O)-G* _(O) A* P = O 10 (21415) A*T*G-CAT-TCT-GCC- P = S61.4 4 3 AAA-AAG-G*A* 11 (21416) A* _(O) T* _(O)G-CAT-TCT- P = S/ 61.7 43 GCC-AAA-AAG_(O)-G* _(O) A* P = O

EXAMPLE 52 NMR experiments on Modified Oligonucleotides Comparison of3′,5′ Versus 2′,5′ Internucleotide Linkages and 2′-substituents Versus3′-substituents by NMR

[0222] The 400 MHz ¹H spectrum of oligomer d(GAU₂*CT), whereU₂*=2′-O-aminohexyluridine showed 8 signals between 7.5 and 9.0 ppmcorresponding to the 8 aromatic protons. In addition, the anomericproton of U* appears as a doublet at 5.9 ppm with J₁′,₂′=7.5 Hz,indicative of C2′-endo sugar puckering. The corresponding 2′-5′ linkedisomer shows a similar structure with J₁′,₂′=3.85 Hz at 5.75 ppm,indicating an RNA type sugar puckering at the novel modification sitefavorable for hybridization to an mRNA target. The proton spectrum ofthe oligomer d(GACU₃*), where U₃*=3′-O-hexylamine, showed the expected 7aromatic proton signals between 7.5 and 9.0 ppm and the anomeric protondoublet at 5.9 ppm with J₁′,₂′=4.4 Hz. This suggests more of a C3′-endopuckering for the 3′-O-alkylamino compounds compared to their 2′analogs. ³¹P NMR of these oligonucleotides showed the expected 4 and 3signals from the internucleotide phosphate linkages for d(GAU*CT) andd(GACU*), respectively. 3′-5′ Linked vs. 2′-5′ linked have differentretention times in RP HPLC and hence different lipophilicities, implyingpotentially different extent of interactions with cell membranes.

EXAMPLE 53 T_(m) Analysis of 2′,5′-Linked Oligonucleotides Versus3′,5′-Linked Oligonucleotides

[0223] Thermal melts were done as per standarized literature procedures.Oligonucleotide identity is as follows: Oligonucleotide A is a normal3′-5′ linked phosphodiester oligodeoxyribonucleotide of the sequenced(GGC TGU* CTG CG) SEQ ID NO: 14 where the * indicates the attachmentsite of a 2′-aminolinker. Oligonucleotide B is a normal 3′-5′ linkedphosphodiester oligoribonucleotide of the sequence d(GGC TGU* CTG CG)SEQ ID NO: 14 where the * indicates the attachment site of a2T-aminolinker. Each of the ribonucleotides of the oligonucleotide,except the one bearing the * substituent, are 2′-O-methylribonucleotides. Oligonucleotide C has 2′-5′ linkage at the * positionin addition to a 3′-aminolinker at this site. The remainder of theoligonucleotide is a phosphodiester oligodeoxyribonucleotide of thesequence d(GGC TGU* CTG CG) SEQ ID NO: 14. The base oligonucleotide (no2′-aminolinker) was not included in the study. TABLE IIIa DNA RNAOLIGONUCLEOTIDE MODIFICATION TARGET TARGET A none 52.2 54.12'-aminolinker 50.9 55.5 B none 51.5 72.3 2'-aminolinker 49.8 69.3 Cnone NA 3'-aminolinker 42.7 51.7

[0224] The 2′-5′ linkages demonstrated a higher melting temperatureagainst an RNA target compared to a DNA target.

EXAMPLE 54 Snake Venom Phosphodiesterase and Liver HomogenateExperiments on Oligonucleotide Stability

[0225] The following oligonucleotides were synthesized following theprocedure of Example 49. TABLE IV Modified Oligonucleotides synthesizedto evaluate stability SEQ ID NO. (ISIS) Back- Che- # # Sequence (5′-3′)bone mistry 15 (22110) TTT-TTT-TTT-TTT-TTT-T*T* P = O 3′-O-MOE T*-T* 16(22111) TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) P = O 3′-O-MOE T ^(#)-U ^(#)15 (22112) TTT-TTT-TTT-TTT-TTT-T*T* P = S 3′-O-MOE T*-T* 16 (22113)TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) P = S 3′-O-MOE T ^(#)-U ^(#) 15(22114) TTT-TTT-TTT-TTT-TTT_(O)-T* P = S/ 3′-O-MOE _(O) T* _(O) T* _(O)T* P = O 16 (22115) TTT-TTT-TTT-TTT-TTT_(O)-T ^(#) P = S/ 3′-O-MOE _(O)T ^(#) _(O) T ^(#) _(O)-U ^(#) P = O

[0226] The oligonucleotides were purified following the procedure ofExample 50 and analyzed for their structure. TABLE V Properties ofModified Oligonucleotides #Ods(260 nm) SEQ ID Expected Observed HPLC²Purified NO.# (ISIS)# #Sequence (5′-3′)¹ Mass Mass T_(R) (min.) 15(22110) TTT-TTT-TTT-TTT-TTT-T*T*T*-T* 6314.189 6315.880 20.39 174 16(22111) TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) T ^(#)-U ^(#) 6004.7775997.490 20.89 147 15 (22112) TTT-TTT-TTT-TTT-TTT-T*T*T*-T* 6298.7996301.730 25.92  224⁻ 16 (22113) TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) T^(#)-U ^(#) 6288.745 6286.940 24.77 209 15 (22114)TTT-TTT-TTT-TTT-TTT_(O)-T* _(O) T* _(O) T* _(O)-T* 6234.799 6237.15024.84 196 16 (22115) TTT-TTT-TTT-TTT-TTT_(O)-T ^(#) _(O) T ^(#) _(O) T^(#) _(O)-U ^(#) 6224.745 6223.780 23.30 340

EXAMPLE 55 3′-O-Aminopropyl modified oligonucleotides

[0227] Following the procedures illustrated above for the synthesis ofoligonucleotides, modified 3′-amidites were used in addition toconventional amidites to prepare the oligonucleotides listed in tablesVI and VII. Nucleosides used include: N6-benzoyl-3′-O-propylphthalimido-A-2′-amidite, 2′-O-propylphthaloyl-A-3′-amidite,2′-O-methoxyethyl-thymidine-3′-amidite (RIC, Inc.),2′-O-MOE-G-3′-amidite (RI Chemical),2′-O-methoxyethyl-5-methylcytidine-3′-amidite,2-O-methoxyethyl-adenosine-3′-amidite (RI Chemical), and5-methylcytidine-3′-amidite. 3′-propylphthalimido-A and2′-propylphthalimido-A were used as the LCA-CPG solid support. Therequired amounts of the amidites were placed in dried vials, dissolvedin acetonitrile (unmodified nucleosides were made into 1M solutions andmodified nucleosides were 100 mg/mL), and connected to the appropriateports on a Millipore Expedite™ Nucleic Acid Synthesis System. Solidsupport resin (60 mg) was used in each column for 2×1 μmole scalesynthesis (2 columns for each oligo were used). The synthesis was runusing the IBP-PS(1 μmole) coupling protocol for phosphorothioatebackbones and CSO-8 for phosphodiesters. The trityl reports indicatednormal coupling results.

[0228] After synthesis the oligonucleotides were deprotected with conc.ammonium hydroxide(aq) containing 10% of a solution of 40% methylamine(aq) at 55° C. for approximately 16 hrs. Then they were evaporated,using a Savant AS160 Automatic SpeedVac, (to remove ammonia) andfiltered to remove the CPG-resin. The crude samples were analyzed by MS,HPLC, and CE. Then they were purified on a Waters 600E HPLC system witha 991 detector using a Waters C4 Prep. scale column (Alice C4 Prep.) andthe following solvents: A: 50 mM TEA-Ac, pH 7.0 and B: acetonitrileutilizing the AMPREP2@ method. After purification the oligonucleotideswere evaporated to dryness and then detritylated with 80% acetic acid atroom temp. for approximately 30 min. Then they were evaporated. Theoligonucleotides were dissolved in conc. ammonium hydroxide and runthrough a column containing Sephadex G-25 using water as the solvent anda Pharmacia LKB SuperFrac fraction collector. The resulting purifiedoligonucleotides were evaporated and analyzed by MS, CE, and HPLC. TABLEVI Oligonucleotides bearing Aminopropyl Substituents SEQ ID NO. (ISIS)Back- # # Sequence (5′-3′)¹ bone 19 (23185-1)A*TG-CAT-TCT-GCC-CCC-AAG-GA* P = S 19 (23186-1) A*TG-CAT-TCT-GCC-CCC-AAG-G A* P = S 20 (23187-1) A* _(O) T _(O) G _(O)-C_(O) A _(S)T_(S)-T_(S)C_(S)T_(S)-G_(S)C_(S)C_(S)-C_(S) P = S/C_(S)C_(S)-A _(O) A _(O) G _(O)-G _(O) A* P = O 20 (23980-1)

_(*O) T _(O) G _(O)-C _(O) A_(S)T_(s)-T_(S)C_(S)T_(S)-G_(S)C_(S)C_(S)-C_(S) P = S/ C_(S)C_(S)-A _(O)A _(O) G _(O)-G

O

* P = O 19 (23981-1)

*TG-CAT-TCT-GCC-CCC-AAG-G

* P = S 19 (23982-1) A*TG-CAT-TCT-GCC-CCC-AAG-GA* P = S

[0229] TABLE VII Aminopropyl Modified Oligonucleotides HPLC CE Re-Expected Observed Retention tention Crude Final Mass Mass Time TimeYield Yield ISIS # (g/mol) (g/mol) (min) (min) (Ods) (Ods) 23185-16612.065 6610.5 23.19 5.98 948 478 23186-1 7204.697 7203.1 24.99 6.181075 379 23187-1 7076.697 7072.33 23.36 7.56 838 546 23980-1 7076.6977102.31 23.42 7.16 984 373 23981-1 7204.697 7230.14 25.36 7.18 1170 52623982-1 6612.065 6635.71 23.47 7.31 1083 463

EXAMPLE 56 In vivo Stability of Modified Oligonucleotides

[0230] The in vivo stability of selected modified oligonucleotidessynthesized in Examples 49 and 55 was determined in BALB/c mice.Following a single i.v. administration of 5 mg/kg of oligonucleotide,blood samples were drawn at various time intervals and analyzed by CGE.For each oligonucleotide tested, 9 male BALB/c mice (Charles River,Wilmington, Mass.) weighing about 25 g were used. Following a one weekacclimatization the mice received a single tail-vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0. One retro-orbital bleed (either at 0.25, 0.5, 2 or 4 hpost-dose) and a terminal bleed (either 1, 3, 8, or 24 h post-dose) werecollected from each group. The terminal bleed (approximately 0.6-0.8 mL)was collected by cardiac puncture following ketamine/xylazineanasthesia. The blood was transferred to an EDTA-coated collection tubeand centrifuged to obtain plasma. At termination, the liver and kidneyswere collected from each mouse. Plasma and tissue homogenates were usedfor analysis to determine intact oligonucleotide content by CGE. Allsamples were immediately frozen on dry ice after collection and storedat −80 C. until analysis.

[0231] The CGE analysis indicated the relative nuclease resistance of2′,5′-linked oligomers compared to ISIS 11061 (Table III, Example 51)(uniformly 2′-deoxy-phosphorothioate oligonucleotide targeted to mousec-raf). Because of the nuclease resistance of the 2′,5′-linkage, coupledwith the fact that 3′-methoxyethoxy substituents are present and affordfurther nuclease protection the oligonucleotides ISIS 17176, ISIS 17177,ISIS 17178, ISIS 17180, ISIS 17181 and ISIS 21415 were found to be morestable in plasma, while ISIS 11061 (Table III) was not. Similarobservations were noted in kidney and liver tissue. This implies that2′,5′-linkages with 3′-methoxyethoxy substituents offer excellentnuclease resistance in plasma, kidney and liver against 5′-exonucleasesand 3′-exonucleases. Thus oligonucleotides with longer durations ofaction can be designed by incorporating both the 2′,5′-linkage and3′-methoxyethoxy motifs into their structure. It was also observed that2′,5′-phosphorothioate linkages are more stable than2′,5′-phosphodiester linkages. A plot of the percentage of full lengtholigonucleotide remaining intact in plasma one hour followingadministration of an i.v. bolus of 5 mg/kg oligonucleotide is shown inFIG. 4.

[0232] A plot of the percentage of full length oligonucleotide remainingintact in tissue 24 hours following administration of an i.v. bolus of 5mg/kg oligonucleotide is shown in FIG. 5.

[0233] CGE traces of test oligonucleotides and a standardphosphorothioate oligonucleotide in both mouse liver samples and mousekidney samples after 24 hours are shown in FIG. 6. As is evident fromthese traces, there is a greater amount of intact oliogonucleotide forthe oligonucleotides of the invention as compared to the standard seenin panel A. The oligonucleotide shown in panel B included onesubstituent of the invention at each of the 5′ and 3′ ends of aphosphorothioate oligonucleotide while the phosphorothioateoligonucleotide seen in panel C included one substituent at the 5′ endand two at the 3′ end. The oligonucleotide of panel D includes asubstituent of the invention incorporated in a 2′,5′ phosphodiesterlinkage at both its 5′ and 3′ ends. While less stable than theoligonucleotide seen in panels B and C, it is more stable than the fullphosphorothioate standard oligonucleotide of panel A.

EXAMPLE 57 Control of c-raf Message in bEND Cells Using ModifiedOligonucleotides

[0234] In order to assess the activity of some of the oligonucleotides,an in vitro cell culture assay was used that measures the cellularlevels of c-raf expression in bEND cells.

[0235] Cells and Reagents

[0236] The bEnd.3 cell line, a brain endothelioma, was obtained from Dr.Werner Risau (Max-Planck Institute). Opti-MEM, trypsin-EDTA and DMEMwith high glucose were purchased from Gibco-BRL (Grand Island, N.Y.).Dulbecco's PBS was purchased from Irvine Scientific (Irvine, Calif.).Sterile, 12 well tissue culture plates and Facsflow solution werepurchased from Becton Dickinson (Mansfield, Mass.). Ultrapureformaldehyde was purchased from Polysciences (Warrington, Pa.). NAP-5columns were purchased from Pharmacia (Uppsala, Sweden).

[0237] Oligonucleotide Treatment

[0238] Cells were grown to approximately 75% confluency in 12 wellplates with DMEM containing 4.5 g/L glucose and 10% FBS. Cells werewashed 3 times with Opti-MEM pre-warmed warmed to 37° C. Oligonucleotidewere premixed with a cationic lipid (Lipofectin reagent, (GIBCO/BRL)and, serially diluted to desired concentrations and transferred on towashed cells for a 4 hour incubation at 37° C. Media was then removedand replaced with normal growth media for 24 hours for northern blotanalysis of mRNA.

[0239] Northern Blot Analysis

[0240] For determination of mRNA levels by Northern blot analysis, totalRNA was prepared from cells by the guanidinium isothiocyanate procedure(Monia et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 15481-15484) 24 hafter initiation of oligonucleotide treatment. Total RNA was isolated bycentrifugation of the cell lysates over a CsCl cushion. Northern blotanalysis, RNA quantitation and normalization to G#PDH mRNA levels weredone according to a reported procedure (Dean and McKay, Proc. Natl.Acad. Sci. USA, 1994, 91, 11762-11766). In bEND cells the2-5-linked-3′-O-methoxyethyl oligonucleotides showed reduction of c-rafmessage activity as a function of concentration. The fact that thesemodified oligonucleotides retained activity promises reduced frequencyof dosing with these oligonucleotides which also show increased in vivonuclease resistance. All 2′,5′-linked oligonucleotides retained theactivity of parent 11061 (Table E) oligonucleotide and improved theactivity even further. A graph of the effect of the oligonucleotides ofthe present invention on c-raf expression (compared to control) in bENDcells is shown in FIG. 7.

EXAMPLE 58 Synthesis of MMI-containing Oligonucleotides

[0241] a. Bis-2′-O-methyl MMI Building Blocks

[0242] The synthesis of MMI (i.e., R=CH₃) dimer building blocks havebeen previously described (see, e.g., Swayze, et al., Synlett 1997, 859;Sanghvi, et al., Nucleosides & Nucleotides 1997, 16 907; Swayze, et al.,Nucleosides & Nucleotides 1997, 16, 971; Dimock, et al., Nucleosides &Nucleotides 1997, 16, 1629). Generally,5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-C-formyl nucleosides werecondensed with 5′-O-(N-methylhydroxylamino)-2′-O-methyl-3′-O-TBDPSnucleosides using 1 equivalent of BH₃ pyridine/1 equivalent ofpyridinium para-toluene sulfonate (PPTS) in 3:1 MeOH/THF. The resultantMMI dimer blocks were then deprotected at the lower part of the sugarwith 15 equivalents of Et₃N-2HF in THF. Thus the T*G^(iBU) dimer unitwas synthesized and phosphitylated to give T*G(MMI)phosphoramidite. In asimilar fashion, A^(BZ)*T(MMI) dimer was synthesized, succinylated andattached to controlled pore glass.

[0243] b. Oligonucleotide Synthesis

[0244] Oligonucleotides were synthesized on a Perseptive BiosystemsExpedite 8901 Nucleic Acid Synthesis System. Multiple 1-μmol syntheseswere performed for each oligonucleotide. A*_(MMI)T solid support wasloaded into the column. Trityl groups were removed with trichloroaceticacid (975 μL over one minute) followed by an acetonitrile wash. Theoligonucleotide was built using a modified thioate protocol. Standardamidites were delivered (210 μL) over a 3 minute period in thisprotocol. The T* _(MMI) amidite was double coupled using 210 μL over atotal of 20 minutes. The amount of oxidizer,3H-1,2-benzodithiole-3-one-1,1-dioxide (Beaucage reagent, 3.4 g Beaucagereagent/200 mL acetonitrile), was 225 μL (one minute wait step). Theunreacted nucleoside was capped with a 50:50 mixture oftetrahydrofuran/acetic anhydride and tetrahydrofuran/pyridine/1-methylimidazole. Trityl yields were followed by the trityl monitor during theduration of the synthesis. The final DMT group was left intact. Afterthe synthesis, the contents of the synthesis cartridge (1 μmole) weretransferred to a Pyrex vial and the oligonucleotide was cleaved from thecontrolled pore glass (CPG) using 5 mL of 30% ammonium hydroxide (NH₄OH)for approximately 16 hours at 55° C.

[0245] c. Oligonucleotide Purification

[0246] After the deprotection step, the samples were filtered from CPGusing Gelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH wasevaporated away in a Savant AS160 automatic SpeedVac. The crude yieldwas measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples were then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer. Trityl-onoligonucleotides were purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions were asfollows: Waters 600E with 991 detector; Waters Delta Pak C4 column(7.8×300 mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH7.0; B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% B forfirst five minutes with linear increase in B to 60% during the next 55minutes. Fractions containing the desired product (retention time=41min. for DMT-ON-16314; retention time 42.5 min. for DMT-ON-16315) werecollected and the solvent was dried off in the SpeedVac.Oligonucleotides were detritylated in 80% acetic acid for approximately60 minutes and lyophilized again. Free trityl and excess salt wereremoved by passing detritylated oligonucleotides through Sephadex G-25(size exclusion chromatography) and collecting appropriate samplesthrough a Pharmacia fraction collector. The solvent was again evaporatedaway in a SpeedVac. Purified oligonucleotides were then analyzed forpurity by CGE, HPLC (flow rate: 1.5 mL/min; Waters Delta Pak C4 column,3.9×300 mm), and MS. The final yield was determined by spectrophotometerat 260 nm.

[0247] The synthesized oligonucleotides and their physicalcharacteristics are shown, respectively, in Tables VIII and IX. Allnucleosides with an asterisk contain MMI linkage. TABLE VIII ICAM-1Oligonucleotides Containing MMI Dimers Synthesized for in Vivo Nucleaseand Pharmacology Studies. SEQ ID NO.# (ISIS)# Sequence (5′-3′) Backbone2′-Chemistry 21 (16134) TGC ATC CCC CAG GCC ACC P = S, MMIBis-2′-OMe-MMI, A*T 2′-H 22 (16315) T*GC ATC CCC CAG GCC P = S, MMIBis-2′-OMe-MMI, ACCA*T2′-H 23 (3082) TGC ATC CCC CAG GCG ACC P = S 2′-H,single AT mismatch 23 (13001) TGC ATC CCC CAG GCC ACC P = S 2′-H AT

[0248] TABLE IX Physical Characteristics of MMI Oligomers Synthesizedfor Pharmacology, and In Vivo Nuclease Studies SEQ ID Observed HPLC NO.(ISIS) Sequence (5′-3′) Mass Time # # Expected Mass (g) (g) (min) Retn.21 (16314) TGC ATC CCC CAG 6295 6297 23.9 GCC ACC A*T 22 (16315) T*G CATC CCC CAG 6302 6303 24.75 GCC ACC A*T

EXAMPLE 59 Synthesis of Sp Terminal Oligonucleotide

[0249] a. 3′-O-t-Butyldiphenylsilyl-thymidine (1)

[0250] 5′-O-Dimethoxytritylthymidine is silylated with 1 equivalent oft-butyldiphenylsilyl chloride (TBDPSC1) and 2 equivalents of imidazolein DMF solvent at room temperature. The 5′-protecting group is removedby treating with 3% dichloracetic acid in CH₂Cl.

[0251] b. 5′-O-Dimethoxytrityl-thymidin-3′-O-yl-N,N-diisopropylamino(S-pivaloyl -2-mercaptoethoxy)phosphoramidite (2)

[0252] 5′-O-Dimethoxytrityl thymidine is treated withbis-(N,N-diisopropylamino)-S-pivaloyl -2-mercaptoethoxy phosphoramiditeand tetrazole in CH₂Cl₂/CH₃CN as described by Guzaev et al., Bioorganic& Medicinal Chemistry Letters 1998, 8, 1123) to yield the titlecompound.

[0253] c.5′-O-Dimethoxytrityl-2′-deoxy-adenosin-3′-O-yl-N,N-diisopropylamino(S-pivaloyl -2-mercapto ethoxy)phosphoramidite (3)

[0254] 5′-O-Dimethoxytrityl-N-6-benzoyl-2′-deoxy-adenosine isphosphitylated as in the previous example to yield the desired amidite.

[0255] d. 3′-O-t-Butyldiphenylsilyl-2′-deoxy-N₂-isobutyryl-guanosine (4)

[0256] 5′-O-Dimethoxytrityl-2′-deoxy-N₂-isobutyryl-guanisine issilylated with TBDPSCl and imidazole in DMF. The 5′-DMT is then removedwith 3% DCA in CH₂Cl₂.

[0257] e. T_((Sp))G dimers and T_((S)) Phosphoramidite

[0258] Compounds 4 and 2 are condensed (1:1 equivalents) using1H-tetrazole in CH₃CN solvent followed by sulfuirization employingBeaucage reagent (see, e.g. Iyer, et al., J. Org. Chem. 1990, 55, 4693).The dimers (TG) are separated by column chromatography and the silylgroup is deprotected using t-butyl ammonium fluoride/THF to give Rp andSp dimers of T_(s)G. Small amounts of these dimers are completelydeprotected and treated with either P1 nuclease or snake venomphosphodiesterase. The R isomer is resistant to P1 nuclease andhydrolyzed by SVPD. The S isomer is resistant to SVPD and hydrolyzed P1nuclease. The Sp isomer of the fully protected T_(s)G dimer isphosphitylated to give DMT-T-Sp-G-phosphoramidite.

[0259] f. A_(s)T Dimers and Solid Support Containing A_(SP)T Dimer

[0260] Compounds 3 and 1 are condensed using 1 H-tetrazole in CH₃CNsolvent followed by sulfuirization to give AT dimers. The dimers areseparated by column chromatography and the silyl group is deprotectedwith TBAF/THF. The configurational assignments are done generally as inthe previous example. The Sp isomer is then attached to controlled poreglass according to standard procedures to give DMT-Asp-T-CPGoligomerization with chirally pure Sp dimer units at the termini.

[0261] g. Oligonucleotide Synthesis

[0262] The oligonucleotide having the sequence T*GC ATC CCC CAG GCC ACCA*T SEQ ID NO: 22 is synthesized, where T*G and A*T represent chiral Spdimer blocks described above. DMT-A_(SP)-T-CPG is taken in the synthesiscolumn and the next 16b residues are built using standardphosphorothioate protocols and 3H-1,2-benzodithiol-3-one 1,1 dioxide asthe sulfurizing agent. After building this 18 mer unit followed by finaldetritylation, the chiral Sp dimer phosphoramidite of 5′-DMT-T_(SP)-Gamidite is coupled to give the desired antisense oligonucleotide. Thiscompound is then deprotected in 30% NH₄OH over 16 hours and the oligomerpurified in HPLC and desalted in Sephader G-25 column. The finaloligomer has Sp configuration at the 5′-terminus and 3′-terminus and theinterior has diastereomeric mixture of Rp and Sp configurations.

EXAMPLE 60 Evaluation of in Vivo Stability of MMI CappedOligonucleotides Mouse Experiment Procedures

[0263] For each oligonucleotide tested, 9 male BALB/c mice (CharlesRiver, Wilmington, Mass.), weighing about 25 g was used (Crooke et al.,J. Pharmacol. Exp. Ther., 1996, 277, 923). Following a 1-weekacclimation, mice received a single tail vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0 One retro-orbital bleed (either 0.25, 0.5, 2 or 4 lv postdose) and a terminal bleed (either 1, 3, 8 or 24 h post dose) werecollected from each group. The terminal bleed (approximately 0.6-0.8 mL)was collected by cardiac puncture following ketamine/xylazineanesthesia. The blood was transferred to an EDTA-coated collection tubeand centrifuged to obtain plasma. At termination, the liver and kidneyswere collected from each mouse. Plasma and tissues homogenates were usedfor analysis for determination of intact oligonucleotide content by CGE.All samples were immediately frozen on dry ice after collection andstored at −80° C. until analysis.

[0264] The capillary gel electrophoretic analysis indicated the relativenuclease resistance of MMI capped oligomers compared to ISIS 3082(uniform 2′-deoxy phosphorothioate). Because of the resistance of MMIlinkage to nucleases, the compound 16314 was found to be stable inplasma while 3082 was not. However, in kidney and liver, the compound16314 also showed certain amount of degradation. This implied that while3′-exonuclease is important in plasma, 5′-exonucleases or endonucleasesmay be active in tissues. To distinguish between these twopossibilities, the data from 16315 was analyzed. In plasma as well as intissues, (liver and kidney) the compound was stable in various timepoints. (1, 3 and 24 hrs.). The fact that no degradation was detectedproved that 5′-exonucleases and 3′-exonuclease are prevalent in tissuesand endonucleases are not active. Furthermore, a single linkage (MMI orSp thioate linkage) is sufficient as a gatekeeper against nucleases.

[0265] Control of ICAM-1 Expression Cells and Reagents

[0266] The bEnd.3 cell line, a brain endothelioma, was the kind gift ofDr. Werner Risau (Max-Planck Institute). Opti-MEM, trypsin-EDTA and DMEMwith high glucose were purchased from Gibco-BRL (Grand Island, N.Y.).Dulbecco's PBS was purchased from Irvine Scientific (Irvine, Calif.).Sterile, 12 well tissue culture plates and Facsflow solution werepurchased from Becton Dickinson (Mansfield, Mass.). Ultrapureformaldehyde was purchased from Polysciences (Warrington, Pa.).Recombinant human TNF-a was purchased from R&D Systems (Minneapolis,Minn.). Mouse interferon-γ was purchased from Genzyme (Cambridge,Mass.). Fraction V, BSA was purchased from Sigma (St. Louis, Mo.). Themouse ICAM-1-PE, VCAM-1-FITC, hamster IgG-FITC and rat IgG₂a-PEantibodies were purchased from Pharmingen (San Diego, Calif.).Zeta-Probe nylon blotting membrane was purchased from Bio-Rad (Richmond,Calif.). QuickHyb solution was purchased from Stratagene (La Jolla,Calif.). A cDNA labeling kit, Prime-a-Gene, was purchased from ProMega(Madison, Wis.). NAP-5 columns were purchased from Pharmacia (Uppsala,Sweden).

[0267] Oligonucleotide Treatment

[0268] Cells were grown to approximately 75% confluency in 12 wellplates with DMEM containing 4.5 g/L glucose and 10% FBS. Cells werewashed 3 times with Opti-MEM pre-warmed to 37° C. Oligonucleotide waspremixed with Opti-MEM, serially diluted to desired concentrations andtransferred onto washed cells for a 4 hour incubation at 37° C. Mediawas removed and replaced with normal growth media with or without 5ng/mL TNF-α and 200 U/mL interferon-γ, incubated for 2 hours fornorthern blot analysis of mRNA or overnight for flow cytometric analysisof cell surface protein expression.

[0269] Flow Cytometry

[0270] After oligonucleotide treatment, cells were detached from theplates with a short treatment of trypsin-EDTA (1-2 min.). Cells weretransferred to 12×75 mm polystyrene tubes and washed with 2% BSA, 0.2%sodium azide in D-PBS at 4° C. Cells were centrifuged at 1000 rpm in aBeckman GPR centrifuge and the supernatant was then decanted. ICAM-1,VCAM-1 and the control antibodies were added at 1 ug/mL in 0.3 mL of theabove buffer. Antibodies were incubated with the cells for 30 minutes at4° C. in the dark, under gentle agitation. Cells were washed again asabove and then resuspended in 0.3 mL of FacsFlow buffer with 0.5%ultrapure formaldehyde. Cells were analyzed on a Becton DickinsonFACScan. Results are expressed as percentage of control expression,which was calculated as follows: [((CAM expression foroligonucleotide-treated cytokine induced cells)—(basal CAMexpression))/((cytokine-induced CAM expression)—(basal CAMexpression))]×100. For the experiments involving cationic lipids, bothbasal and cytokine-treated control cells were pretreated with Lipofectinfor 4 hours in the absence of oligonucleotides.

[0271] The results reveal the following: 1) Isis 3082 showed an expecteddose response (25-200 nM); 2) Isis 13001 lost its ability to inhibitICAM-1 expression as expected from a mismatch compound, thus proving anantisense mechanism; 3) 3′-MMI capped oligomer 16314 improved theactivity of 3082, and at 200 nM concentration, nearly twice as active as3082; 4) 5′- and 3′-MMI capped oligomer is the most potent compound andit is nearly 4 to 5 times more efficacious than the parent compound at100 and 200 nM concentrations. Thus, improved nuclease resistanceincreased the potency of the antisense oligonucleotides.

EXAMPLE 61 Control of H-ras Expression

[0272] Antisense oligonucleotides targeting the H-ras message weretested for their ability to inhibit production of H-ras mRNA in T-24cells. For these test, T-24 cells were plated in 6-well plates and thentreated with various escalating concentrations of oligonucleotide in thepresence of cationic lipid (Lipofectin, GIBCO) at the ratio of 2.5 μg/mlLipofectin per 100 nM oligonucleotide. Oligonucleotide treatment wascarried out in serum free media for 4 hours. Eighteen hours aftertreatment the total RNA was harvested and analyzed by northern blot forH-ras mRNA and control gene G3PDH. The data is presented in FIGS. 8 and9 in bar graphs as percent control normalized for the G3PDH signal. Ascan be seen, the oligonucleotide having a single MMI linkage in each ofthe flank regions showed significant reduction of H-ras mRNA.

EXAMPLE 62 5-Lipoxygenase Analysis and Assays

[0273] A. Therapeutics

[0274] For therapeutic use, an animal suspected of having a diseasecharacterized by excessive or abnormal supply of 5-lipoxygenase istreated by administering a compound of the invention. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Such treatment is generallycontinued until either a cure is effected or a diminution in thediseased state is achieved. Long term treatment is likely for somediseases.

[0275] B. Research Reagents

[0276] The oligonucleotides of the invention will also be useful asresearch reagents when used to cleave or otherwise modulate5-lipoxygenase mRNA in crude cell lysates or in partially purified orwholly purified RNA preparations. This application of the invention isaccomplished, for example, by lysing cells by standard methods,optimally extracting the RNA and then treating it with a composition atconcentrations ranging, for instance, from about 100 to about 500 ng per10 Mg of total RNA in a buffer consisting, for example, of 50 mmphosphate, pH ranging from about 4-10 at a temperature from about 30 toabout 50° C. The cleaved 5-lipoxygenase RNA can be analyzed by agarosegel electrophoresis and hybridization with radiolabeled DNA probes or byother standard methods.

[0277] C. Diagnostics

[0278] The oligonucleotides of the invention will also be useful indiagnostic applications, particularly for the determination of theexpression of specific mRNA species in various tissues or the expressionof abnormal or mutant RNA species. In this example, while themacromolecules target a abnormal mRNA by being designed complementary tothe abnormal sequence, they would not hybridize to normal mRNA. Tissuesamples can be homogenized, and RNA extracted by standard methods. Thecrude homogenate or extract can be treated for example to effectcleavage of the target RNA. The product can then be hybridized to asolid support which contains a bound oligonucleotide complementary to aregion on the 5′ side of the cleavage site. Both the normal and abnormal5′ region of the mRNA would bind to the solid support. The 3′ region ofthe abnormal RNA, which is cleaved, would not be bound to the supportand therefore would be separated from the normal mRNA.

[0279] Targeted mRNA species for modulation relates to 5-lipoxygenase;however, persons of ordinary skill in the art will appreciate that thepresent invention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

[0280] D. In Vitro Assays

[0281] The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of oligonucleotides which interfere with5-lipoxygenase synthesis in these cells.

[0282] A second test system for oligonucleotides makes use of the factthat 5-lipoxygenase is a “suicide” enzyme in that it inactivates itselfupon reacting with substrate. Treatment of differentiated HL-60 or othercells expressing 5 lipoxygenase, with 10 μM A23187, a calcium ionophore,promotes translocation of 5-lipoxygenase from the cytosol to themembrane with subsequent activation of the enzyme. Following activationand several rounds of catalysis, the enzyme becomes catalyticallyinactive. Thus, treatment of the cells with calcium ionophoreinactivates endogenous 5-lipoxygenase. It takes the cells approximately24 hours to recover from A23187 treatment as measured by their abilityto synthesize leukotriene B₄. Macromolecules directed against5-lipoxygenase can be tested for activity in two HL-60 model systemsusing the following quantitative assays. The assays are described fromthe most direct measurement of inhibition of 5-lipoxygenase proteinsynthesis in intact cells to more downstream events such as measurementof 5-lipoxygenase activity in intact cells.

[0283] A direct effect which oligonucleotides can exert on intact cellsand which can be easily be quantitated is specific inhibition of5-lipoxygenase protein synthesis. To perform this technique, cells canbe labeled with ³⁵S-methionine (50 μCi/mL) for 2 hours at 37° C. tolabel newly synthesized protein. Cells are extracted to solubilize totalcellular proteins and 5-lipoxygenase is immunoprecipitated with5-lipoxygenase antibody followed by elution from A Sepharose beads. Theimmunoprecipitated proteins are resolved by SDS-protein polyacrylamidegel electrophoresis and exposed for autoradiography. The amount ofimmunoprecipitated 5-lipoxygenase is quantitated by scanningdensitometry.

[0284] A predicted result from these experiments would be as follows.The amount of 5-lipoxygenase protein immunoprecipitated from controlcells would be normalized to 100%. Treatment of the cells with 1 μM, 10μM, and 30 μM of the macromolecules of the invention for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control,respectively.

[0285] Measurement of 5-lipoxygenase enzyme activity in cellularhomogenates could also be used to quantitate the amount of enzymepresent which is capable of synthesizing leukotrienes. A radiometricassay has now been developed for quantitating 5-lipoxygenase enzymeactivity in cell homogenates using reverse phase HPLC. Cells are brokenby sonication in a buffer containing protease inhibitors and EDTA. Thecell homogenate is centrifuged at 10,000×g for 30 min and thesupernatants analyzed for 5-lipoxygenase activity. Cytosolic proteinsare incubated with 10 μM ¹⁴C-arachidonic acid, 2 mM ATP, 50 μM freecalcium, 100 μg/mL phosphatidylcholine, and 50 mM bis-Tris buffer, pH7.0, for 5 min at 37° C. The reactions are quenched by the addition ofan equal volume of acetone and the fatty acids extracted with ethylacetate. The substrate and reaction products are separated by reversephase HPLC on a Novapak C18 column (Waters Inc., Millford, Mass.).Radioactive peaks are detected by a Beckman model 171radiochromatography detector. The amount of arachidonic acid convertedinto di-HETE's and mono-HETE's is used as a measure of 5-lipoxygenaseactivity.

[0286] A predicted result for treatment of DMSO differentiated HL-60cells for 72 hours with effective the macromolecules of the invention at1 μM, 10 μM, and 30 μM would be as follows. Control cells oxidize 200pmol arachidonic acid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM,and 30 μM of an effective oligonucleotide would oxidize 195 pmol, 140pmol, and 60 pmol of arachidonic acid/5 min/10⁶ cells respectively.

[0287] A quantitative competitive enzyme linked immunosorbant assay(ELISA) for the measurement of total 5-lipoxygenase protein in cells hasbeen developed. Human 5-lipoxygenase expressed in E. coli and purifiedby extraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC isused as a standard and as the primary antigen to coat microtiter plates.Purified 5-lipoxygenase (25 ng) is bound to the microtiter platesovernight at 4° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM Tris!HCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labeled secondantibody determined by development with tetramethylbenzidine.

[0288] Predicted results from such an assay using a 30 meroligonucleotide at 1 μM, 10 μM, and 30 μM would be 30 ng, 18 ng and 5 ngof 5-lipoxygenase per 10⁶ cells, respectively with untreated cellscontaining about 34 ng 5-lipoxygenase.

[0289] A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophil-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of the macromolecule for 48-72 hours in thepresence of 1.3% DMSO. The cells are washed and resuspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining-1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

[0290] Using this assay the following results would likely be obtainedwith an oligonucleotide directed to the 5-LO mRNA. Cells will be treatedfor 72 hours with either 1 μM, 10 μM or 30 μM of the macromolecule inthe presence of 1.3% DMSO. The quantity of LTB₄ produced from 5×10⁵cells would be expected to be about 75 pg, 50 pg, and 35 pg,respectively with untreated differentiated cells producing 75 pg LTB₄.

[0291] E. In Vivo Assay

[0292] Inhibition of the production of 5-lipoxygenase in the mouse canbe demonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oligonucleotides will be applied topically to both ears 12 to 24hours prior to administration of arachidonic acid to allow optimalactivity of the compounds. Both ears are pretreated for 24 hours witheither 0.1 μmol, 0.3 μmol, or 1.0 μmol of the macromolecule prior tochallenge with arachidonic acid. Values are expressed as the mean forthree animals per concentration. Inhibition of polymorphonuclear cellinfiltration for 0.1 μmol, 0.3 μmol, and 1 μmol is expected to be about10%, 75% and 92% of control activity, respectively. Inhibition of edemais expected to be about 3%, 58% and 90%, respectively while inhibitionof leukotriene B₄ production would be expected to be about 15%, 79% and99%, respectively.

EXAMPLE 63 5′-O-DMT-2′-deoxy-2′-methylene5-methyluridine-3′-(2-cyanoethyl-N,N-diisoproppyl) phosphoramidite

[0293] 2′-Deoxy-2′-methylene-3 ′,5′-O-(tetraisopropyldisiloxane-1,3,diyl)-5-methyl uridine is synthesized following theprocedures reported for the corresponding uridine derivative (Hansske,F.; Madej, D.; Robins, M. J. Tetrahedron (1984) 40, 125; Matsuda, A.;Takenusi, K.; Tanaka, S.; Sasaki, T.; Ueda, T. J. Med. Chem. (1991) 34,812; See also Cory, A. H.; Samano, V.; Robins, M. J.; Cory, J. G.2′-Deoxy-2′-methylene derivatives of adenosine, guanosine, tubercidin,cytidine and uridine as inhibitors of L1210 cell growth in culture.Biochem. Pharmacol. (1994), 47(2), 365-71.)

[0294] It is treated with IM TBAF in THF to give2′-deoxy-2′-methylene-5-methyl uridine. It is dissolved in pyridine andtreated with DMT-Cl and stirred to give the5′-O-DMT-2′-deoxy-2′-methylene-5-methyl uridine. This compound istreated with 2-cyanoethyl-N,N-diisopropyl phosphoramidite anddiisopropylaminotetrazolide. In a similar manner the corresponding N-6benzoyl adenosine, N-4-benzoyl cytosine, N-2-isobutyryl guanosinephosphoramidite derivatives are synthesized.

EXAMPLE 63 Synthesis of 3′-O-4′-C-methyleneribonucleoside

[0295] 5′-O-DMT-3′-O-4′-C-methylene uridine and 5-methyl uridine aresynthesized and phosphitylated according to the procedure of Obika etal. (Obika et al. Bioorg. Med. Chem. Lett. (1999) 9, 515-158). Theamidites are incorporated into oligonucleotides using the protocolsdescribed above.

EXAMPLE 64 Synthesis of 2′-methylene phosphoramidites

[0296]5′-O-DMT-2′-(methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite,5′-O-DMT-2′-(methyl)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-(methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-(methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites wereobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides were synthesized according to the procedure described byIribarren, Adolfo M.; Cicero, Daniel O.; Neuner, Philippe J. Resistanceto degradation by nucleases of(2′S)-2′-deoxy-2′-C-methyloligonucleotides, novel potential antisenseprobes. Antisense Res. Dev., (1994), 4(2), 95-8; Schmit, Chantal;Bevierre, Marc-Olivier; De Mesmaeker, Alain; Altmann, Karl-Heinz. “Theeffects of 2′- and 3′-alkyl substituents on oligonucleotidehybridization and stability”. Bioorg. Med. Chem. Lett. (1994), 4(16),1969-74.

[0297] The phosphitylation is carried out by using the bisamiditeprocedure.

EXAMPLE 65 Synthesis of 2′-S-methyl phosphoramidites

[0298]5′-O-DMT-2′-S-(methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-S(methyl)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-S-(methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-S-(methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites wereobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides were synthesized according to the procedure described byFraser et al. (Fraser, A.; Wheeler, P.; Cook, P. D.; Sanghvi, Y. S. J.Heterocycl. Chem. (1993) 31, 1277-1287). The phosphitylation is carriedout by using the bisamidite procedure.

EXAMPLE 66 Synthesis of 2′-O-methyl-β-D-arabinofuranosyl compounds

[0299] 2′-O-Methyl-β-D-arabinofuranosyl-thymidine containingoligonucleotides were synthesized following the procedures of Gotfredsonet. al. (Gotfredson, C. H. et. al. Tetrahedron Lett. (1994) 35,6941-6944; Gotfredson, C. H. et. al. Bioorg. Med. Chem. (1996) 4,1217-1225).5′-O-DMT-2′-ara-(O-methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(O-methyl)-N-6-benzoyladenosine (3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-ara-(O-methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT -2′-ara-(O-methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byGotfredson, C. H. et. al. Tetrahedron Lett. (1994) 35, 6941-6944;Gotfredson, C. H. et. al. Bioorg. Med. Chem. (1996) 4, 1217-1225. Thephosphitylation is carried out by using the bisamidite procedure.

EXAMPLE 67 Synthesis of 2′-fluoro-β-D-arabinofuranosyl compounds

[0300] 2′-Fluoro-β-D-arabinofuranosyl oligonucleotides are synthesizedfollowing the procedures by Kois, P. et al., Nucleosides Nucleotides 12,1093,1993 and Darnha et al., J. Am. Chem. Soc., 120, 12976, 1998 andreferences sited therin.5′-O-DMT-2′-ara-(fluoro)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT -2′-ara-(fluoro)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-ara-(fluoro)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and5′-O-DMT-2′-ara-(fluoro)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byKois, P. et al., Nucleosides Nucleotides 12, 1093, 1993 and Damha etal., J. Am. Chem. Soc., 120, 12976, 1998. The phosphitylation is carriedout by using the bisamidite procedure.

EXAMPLE 68 Synthesis of 2′-hydroxyl-β-D-arabinofuranosyl compounds

[0301]2′-Hydroxyl-β-D-arabinofuranosyl oligonucleotides are synthesizedfollowing the procedures by Resmini and Pfleiderer Helv. Chim. Acta, 76,158,1993; Schmit et al., Bioorg. Med. Chem. Lett. 4, 1969, 1994 Resmini,M.; Pfleiderer, W. Synthesis of arabinonucleic acid (tANA). Bioorg. Med.Chem. Lett. (1994), 16, 1910.; Resmini, Matthias; Pfleiderer, W.Nucleosides. Part LV. Efficient synthesis of arabinoguanosine buildingblocks (Helv. Chim. Acta, (1994), 77, 429-34; and Damha et al., J. Am.Chem. Soc., 1998, 120, 12976, and references sited therin).

[0302]5′-O-DMT-2-ara-(hydroxy)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(hydroxy)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-ara-(hydroxy)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-ara-(hydroxy) -N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphorarnidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byKois, P. et al., Nucleosides Nucleotides 12, 1093, 1993 and Damha etal., J. Am. Chem. Soc., 120, 12976, 1998. The phosphitylation is carriedout by using the bisamidite procedure.

EXAMPLE 69 Synthesis of difluoromethylene compounds

[0303]5′-O-DMT-2′-deoxy-2′-difluoromethylene-5-methyluridine-3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite), 5′-O-DMT-2′-deoxy-2′-difluoromethylene-N-4-benzoyl-cytidine,5′-O-DMT-2′-deoxy-2′-diflyoromethylene-N-6-benzoyl adenosine, and5′-O-DMT -2′-deoxy-2′-difluoroethylene-N₂-isobutyryl guanosine aresynthesized following the protocols described by Usman et. al. (U.S.Pat. No. 5,639,649, Jun. 17, 1997).

EXAMPLE 70 Synthesis of5′-O-DMT-2′-deoxy-2′-β-(O-acetyl)-21-α-methyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropyl phosphoramidite

[0304] 5′-O-DMT-2′-deoxy-2′-β-(OH)-2′-α-methyl-adenosine is synthesizedfrom the compound 5′-3′-protected-2′-keto-adenosine (Rosenthal, Sprinzland Baker, Tetrahedron Lett. 4233, 1970; see also Nucleic acid relatedcompounds. A convenient procedure for the synthesis of 2′- and3′-ketonucleosides is shown Hansske et al., Dep. Chem., Univ. Alberta,Edmonton, Can., Tetrahedron Lett. (1983), 24(15), 1589-92.) by Grigandaddition of MeMgI in THF solvent, followed by seperation of the isomers.The 2-β-(OH) is protected as acetate. 5′-3′-acetal group is removed,5′-position dimethoxy, tritylated, N-6 position is benzoylated and then3′-position is phosphitylated to give5′-O-DMT-2′-deoxy-2′-β-(O-acetyl)-2′-α-methyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite.

EXAMPLE 71 Synthesis of5′-O-DMT-2′-α-ethynyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite

[0305] 5′-O-DMT-2′-deoxy-2′-β-(OH)-2′-α-ethynyl-adenosine is synthesizedfrom the compound 5′-3′-protected-2′-keto-adenosine (Rosenthal, Sprinzland Baker, Tetrahedron Lett. 4233, 1970) by Grigand addition ofEthynyl-MgI in THF solvent, followed by seperation of the isomers. The2′-β-(OH) is removed by Robins' deoxygenation procedure (Robins et al.,J. Am. Chem. Soc. (1983), 105, 4059-65. 5′-3′-acetal group is removed,5′-position dimethoxytritylated, N-6 position is benzoylated and then3′-position is phosphitylated to give the title compound.

EXAMPLE 72 2′-O-(guaiacolyl)-5-methyluridine

[0306] 2-Methoxyphenol (6.2 g, 50 mmol) was slowly added to a solutionof borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a100 mL bomb. Hydrogen gas evolved as the solid dissolvedO-2,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate(2.5 mg) were added and the bomb was sealed, placed in an oil bath andheated to 155° C. for 36 hours. The bomb was cooled to room temperatureand opened. The crude solution was concentrated and the residuepartitioned between water (200 mL) and hexanes (200 mL). The excessphenol was extracted into hexanes. The aqueous layer was extracted withethyl acetate (3×200 mL) and the combined organic layer was washed oncewith water, dried over anhydrous sodium sulfate and concentrated. Theresidue was purified by silica gel flash column chromatography usingmethanol:methylene chloride (1/10, v/v) as the eluent. Fractions werecollected and the target fractions were concentrated to give 490 mg ofpure product as a white-solid. Rf=0.545 in CH₂Cl₂/CH₃H (10:1). MS/ES forC₁₇H₂ON₂O₇,364.4; Observed 364.9.

EXAMPLE 735′-Dimethoxytrityl-2′-O-(2-methoxyphenyl)-5-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite

[0307] 2′-O-(guaiacolyl)-5-methyl-uridine is treated with 1.2equivalents of dimethoxytrityl chloride (DMT-Cl) in pyridine to yieldthe 5′-O-dimethoxy tritylated nucleoside. After evaporation of thepyridine and work up (CH₂Cl₂/saturated NaHCO₃ solution) the compound ispurified in a silica gel column. The 5′-protected nucleoside isdissolved in anhydrous methylene chloride and under argon atmosphere,N,N-diisopropylaminohydro-tetrazolide (0.5 equivalents) andbis-N,N-diisopropylamino-2-cyanoethyl-phosphoramidite (2 equivalents)are added via syringe over 1 min. The reaction mixture is stirred underargon at room temperature for 16 hours and then applied to a silicacolumn. Elution with hexane:ethylacetate (25:75) gives the titlecompound.

EXAMPLE 745′-Dimethoxytrityl-2′-O-(2-methoxyphenyl)-5-methyluridine-3′-O-succinate

[0308] The 5′-protected nucleoside from Example 73 is treated with 2equivalents of succinic anhydride and 0.2 equivalents of4-N,N-dimethylaminopyridine in pyridine. After 2 hours the pyridine isevaporated, the residue is dissolved in CH₂Cl₂ and washed three timeswith 100 mL of 10% citric acid solution. The organic layer is dried overanhydrous MgSO₄ to give the desired succinate. The succinate is thenattached to controlled pore glass (CPG) using established procedures(Pon, R. T., Solid phase supports for oligonucleotide synthesis, inProtocols for Oligonucleotides and Analogs, S. Agrawal (Ed.), HumanaPress: Totawa, N.J., 1993, 465-496).

EXAMPLE 75 5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine

[0309] 2′-3′-O-Dibutylstannyl-5-methyl uridine (Wagner et al., J. Org.Chem., 1974, 39, 24) is alkylated with trans-2-methoxycyclohexyltosylate at 70° C. in DMF. A 1:1 mixture of 2′-O— and3′-O-(trans-2-methoxycyclohexyl)-5-methyluridine is obtained in thisreaction. After evaporation of the DMF solvent, the crude mixture isdissolved in pyridine and treated with dimethoxytritylchloride (DMT-Cl)(1.5 equivalents). The resultant mixture is purified by silica gel flashcolumn chromatography to give the title compound.

EXAMPLE 765′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite

[0310] 5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine is phosphitylated according to the procedure described above togive the required phosphoramidite.

EXAMPLE 775′-Dimethoxytrityl-2′-O-(trans-2-metboxycyclohexyl)-5-methyluridine-3′-O-(succinyl-amino) CPG

[0311] 5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine is succinylated and attached to controlled pore glass to givethe solid support bound nucleoside.

EXAMPLE 78 trans-2-ureido-cyclohexanol

[0312] Trans-2-amino-cyclohexanol (Aldrich) is treated with triphosgenein methylene chloride (1/3 equivalent). To the resulting solution,excess ammonium hydroxide is added to give after work up the titlecompound.

EXAMPLE 79 2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridine

[0313] Trans-2-uriedo-cyclohexanol (50 mmol) is added to a solution ofborane in tetrahydrofuran (1 M, 10 mL, 10 mmol) while stirring in a 10mL bomb. Hydrogen gas evolves as the reactant dissolves.O2,2′-Anhydro-5-methyluridine (5 mmol) and sodium bicarbonate (2.5 mg)are added to the bomb and sealed. Then it is heated to 140 for 72 hrs.The bomb is cooled to room temperature and opened. The crude materialwas worked up as illustrated above followed by purification by silicagel flash column chromatography to give the title compound.

EXAMPLE 805′-O-(Dimethoxytrityl)2′-O-(trans-2-uriedo-cyclohexyl)3′-O-(2-cyanoethyl,N,N,-diisopropyl)uridine phosphoramidite

[0314] 2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridine tritylated atthe 5′-OH and phosphitylated at the 3′-OH following the proceduresillustrated in example 2 to give the title compound.

EXAMPLE 815′-O-dimethoxytrityl-2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl-3′-O-(succinyl)-aminoCPG uridine

[0315] 5′-O-dimethoxytrityl-2′-O-(trans-2-uriedo-cyclohexyl)-5-methyluridine is succinylated and attached to CPG as illustrated above.

EXAMPLE 82 2′-O-(trans-2-methoxy-cyclohexyl) adenosine

[0316] Trans-2-methoxycyclopentanol, trans-2-methoxycylcohexanol,trans-2-methoxy-cyclopentyl tosylate and trans-2-methoxy-cyclohexyltosylate are prepared according to reported procedures (Roberts, D. D.,Hendrickson, W., J. Org. Chem., 1967, 34, 2415-2417; J. Org. Chem.,1997, 62, 1857-1859). A solution of adenosine (42.74 g, 0.16 mol) in drydimethylformamide (800 mL) at 5° C. is treated with sodium hydride (8.24g, 60% in oil prewashed thrice with hexanes, 0.21 mol). After stirringfor 30 min, trans-2-methoxycyclohexyl tosylate (0.16 mol) is added over20 minutes at 5° C. The reaction is stirred at room temperature for 48hours, then filtered through Celite. The filtrate is concentrated underreduced pressure followed by coevaporation with toluene (2×100 mL) togive the title compound.

EXAMPLE 83 N⁶-Benzoyl-2′-O-(trans-2-methoxycyclohexyl)adenosine

[0317] A solution of 2′-O-(trans-2-methoxy-cyclohexyl)adenosine (0.056mol) in pyridine (100 mL) is evaporated under reduced pressure todryness. The residue is redissolved in pyridine (560 mL) and cooled inan ice water bath. Trimethylsilyl chloride (36.4 mL, 0.291 mol) is addedand the reaction is stirred at 5° C. for 30 minutes. Benzoyl chloride(33.6 mL, 0.291 mol) is added and the reaction is allowed to warm to 25°C. for 2 hours and then cooled to 5° C. The reaction is diluted withcold water (112 mL) and after stirring for 15 min, concentrated ammoniumhydroxide (112 mL). After 30 min, the reaction is concentrated underreduced pressure (below 30° C.) followed by coevaporation with toluene(2×100 mL). The residue is dissolved in ethyl acetate-methanol (400 mL,9:1) and the undesired silyl by-products are removed by filtration. Thefiltrate is concentrated under reduced pressure and purified by silicagel flash column chromatography (800 g, chloroform-methanol 9:1).Selected fractions are combined, concentrated under reduced pressure anddried at 25° C./0.2 mmHg for 2 h to give the title compound.

EXAMPLE 84N⁶-Benzoyl-5′-O-(dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosine

[0318] A solution of N⁶-benzoyl-2′-O-(trans-2-methoxycyclohexyl)adenosine (0.285 mol) in pyridine (100 mL) is evaporated under reducedpressure to an oil. The residue is redissolved in dry pyridine (300 mL)and 4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl, 10.9 g, 95%, 0.31mol) added. The mixture is stirred at 25° C. for 16 h and then pouredonto a solution of sodium bicarbonate (20 g) in ice water (500 mL). Theproduct is extracted with ethyl acetate (2×150 mL). The organic layer iswashed with brine (50 mL), dried over sodium sulfate (powdered) andevaporated under reduced pressure (below 40° C.). The residue ischromato-graphed on silica gel (400 g, ethyl acetate-hexane 1:1.Selected fractions were combined, concentrated under reduced pressureand dried at 25° C./0.2 mmHg to give the title compound.

EXAMPLE 85[N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosine-3′-O-yl]-N,N-diisopropylamino-cyanoethoxyphosphoramidite

[0319] Phosphitylation ofN⁶-benzoyl-5′-O-(dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosinewas performed as illustrated above to give the title compound.

EXAMPLE 86 General procedures for chimeric C3′-endo and C2′-endomodified oligonucleotide synthesis

[0320] Oligonucleotides are synthesized on a PerSeptive BiosystemsExpedite 8901 Nucleic Acid Synthesis System. Multiple 1-mmol synthesesare performed for each oligonucleotide. The 3′-end nucleoside containingsolid support is loaded into the column. Trityl groups are removed withtrichloroacetic acid (975 mL over one minute) followed by anacetonitrile wash. The oligonucleotide is built using a modified diester(P═O) or thioate (P═S) protocol.

[0321] Phosphodiester Protocol

[0322] All standard amidites (0.1 M) are coupled over a 1.5 minute timeframe, delivering 105 μL material. All novel amidites are dissolved indry acetonitrile (100 mg of amidite/1 mL acetonitrile) to giveapproximately 0.08-0.1 M solutions. The 2′-modified amidites (both riboand arabino monomers) are double coupled using 210 μL over a total of 5minutes. Total coupling time is approximately 5 minutes (210 mL ofamidite delivered). 1-H-tetrazole in acetonitrile is used as theactivating agent. Excess amidite is washed away with acetonitrile. (1S)-(+)-(10-camphorsulfonyl) oxaziridine (CSO, 1.0 g CSO/8.72 mL dryacetonitrile) is used to oxidize (3 minute wait step) deliveringapproximately 375 μL of oxidizer. Standard amidites are delivered (210μL) over a 3-minute period.

[0323] Phosphorothioate Protocol

[0324] The 2′-modified amidite is double coupled using 210 μL over atotal of 5 minutes. The amount of oxidizer,3H-1,2-benzodithiole-3-one-1,1-dioxide (Beaucage reagent, 3.4 g Beaucagereagent/200 mL acetonitrile), is 225 μL (one minute wait step). Theunreacted nucleoside is capped with a 50:50 mixture oftetrahydrofuran/acetic anhydride and tetrahydrofuran/pyridine/1-methylimidazole. Trityl yields are followed by the trityl monitor during theduration of the synthesis. The final DMT group is left intact. After thesynthesis, the contents of the synthesis cartridge (1 mmole) istransferred to a Pyrex vial and the oligonucleotide is cleaved from thecontrolled pore glass (CPG) using 30% ammonium hydroxide (NH₄OH, 5 mL)for approximately 16 hours at 55° C.

[0325] Oligonucleotide Purification

[0326] After the deprotection step, the samples are filtered from CPGusing Gelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH isevaporated away in a Savant AS160 automatic speed vac. The crude yieldis measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples are then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer. Trityl-onoligonucleotides are purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions are asfollows: Waters 600E with 991 detector; Waters Delta Pak C4 column(7.8×300 mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH7.0; Solvent B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% Bfor first five minutes with linear increase in B to 60% during the next55 minutes. Fractions containing the desired product/s (retentiontime=41 minutes for DMT-ON-16314; retention time=42.5 minutes forDMT-ON-16315) are collected and the solvent is dried off in the speedvac. Oligonucleotides are detritylated in 80% acetic acid forapproximately 60 minutes and lyophilized again. Free trityl and excesssalt are removed by passing detritylated oligonucleotides throughSephadex G-25 (size exclusion chromatography) and collecting appropriatesamples through a Pharmacia fraction collector. The solvent is againevaporated away in a speed vac. Purified oligonucleotides are thenanalyzed for purity by CGE, HPLC (flow rate: 1.5 mL/min; Waters DeltaPak C4 column, 3.9×300 mm), and MS. The final yield is determined byspectrophotometer at 260 nm.

[0327] Procedures

[0328] Procedure 1

[0329] ICAM-1 Expression

[0330] Oligonucleotide Treatment of HUVECs

[0331] Cells were washed three times with Opti-MEM (Life Technologies,Inc.) prewarmed to 37° C. Oligonucleotides were premixed with 10 g/mLLipofectin (Life Technologies, Inc.) in Opti-MEM, serially diluted tothe desired concentrations, and applied to washed cells. Basal anduntreated (no oligonucleotide) control cells were also treated withLipofectin. Cells were incubated for 4 h at 37° C., at which time themedium was removed and replaced with standard growth medium with orwithout 5 mg/mL TNF-α 7 & D Systems). Incubation at 37° C. was continueduntil the indicated times.

[0332] Quantitation of ICAM-1 Protein Expression byFluorescence-activated Cell Sorter

[0333] Cells were removed from plate surfaces by brief trypsinizationwith 0.25% trypsin in PBS. Trypsin activity was quenched with a solutionof 2% bovine serum albumin and 0.2% sodium azide in PBS (+Mg/Ca). Cellswere pelleted by centrifugation (1000 rpm, Beckman GPR centrifuge),resuspended in PBS, and stained with 3 1/10⁵ cells of the ICAM-1specific antibody, CD54-PE (Pharmingin). Antibodies were incubated withthe cells for 30 min at 4 C. in the dark, under gently agitation. Cellswere washed by centrifugation procedures and then resuspended in 0.3 mLof FacsFlow buffer (Becton Dickinson) with 0.5% formaldehyde(Polysciences). Expression of cell surface ICAM-1 was then determined byflow cytometry using a Becton Dickinson FACScan. Percentage of thecontrol ICAM-1 expression was calculated as follows:[(oligonucleotide-treated ICAM-1 value)−(basal ICAM-1value)/(non-treated ICAM-1 value)−(basal ICAM-1 value)]. (Baker, Brenda,et. al. 2′-O-(2-Methoxy)ethyl-modified Anti-intercellular AdhesionMolecule 1 (ICAM-1) Oligonucleotides Selectively Increase the ICAM-1mRNA Level and Inhibit Formation of the ICAM-1 Translation InitiationComplex in Human Umbilical Vein Endothelial Cells, The Journal ofBiological Chemistry, 272, 11994-12000, 1997.)

[0334] ICAM-1 expression of chimeric C3′-endo and C2′-endo modifiedoligonucleotides of the invention is measured by the reduction of ICAM-1levels in treated HUVEC cells. The oligonucleotides are believed to workby RNase H cleavage mechanism. Appropriate scrambled controloligonucleotides are used as controls. They have the same basecomposition as the test sequence.

[0335] Sequences that contain the chimeric C3′-endo (2′-MOE)and C2′-endo(one of the following modifications: 2′-S—Me, 2′-Me, 2′-ara-F,2′-ara-OH, 2′-ara-O-Me) as listed in Table X below are prepared andtested in the above assay. SEQ ID NO: 24, a C-raf targetedoligonucleotide, is used as a control. TABLE X OligonucleotidesContaining chimeric 2′-O-(2-methoxyethyl) and 2′-S-(methyl) modifica-tions. SEQ ID NO: Sequence (5′-3′) Target 24 AsTsGs C ^(m) sAsTsTsCs^(m)Ts GsCs_(m) mouse Cs^(m) Cs^(m)C^(m)sC ^(m) s AsAsGs GsA C-raf25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs human GsGsC^(m)s ASTsC^(m)SC^(m)sGSTs ICAM-1 C^(m)SA

[0336] All nucleosides in bold are 2′-O-(methoxyethyl); subscript sindicates a phosphorothioate linkage; underlined nucleosides indicate2′-S—Me-modification. Superscript m on C (Cm)indicates a 5-methyl-C.TABLE XI Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-O-(methyl) modifica- tions SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA

[0337] All nucleosides in bold are 2′-O-(methoxyethyl); subscript sindicates a phosphorothioate linkage; underlined nucleosides indicate2′-Methyl modification. Superscript m on C (Cm)indicates a 5-methyl-C.TABLE XII Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-ara-(fluoro) modifi- cations SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA --

[0338] All nucleosides in bold are 2′-O-(methoxyethyl); subscript sindicates a phosphorothioate linkage; underlined nucleosides indicate2′-ara-(fluoro) modification. superscript m on C (Cm)indicates a5-methyl-C. TABLE XIII Oligonucleotides Containing chimeric2′-O-(2-methoxyethyl) and 2′-ara-(OH) modifica- tions SEQ ID NO:Sequence (5′-3′) Target 24 AsTsGs C^(m)sAsTs TsCs^(m)Ts mouseGsCs^(m)Cs^(m)Cs^(m)C^(m)sC^(m)s AsAsGs C-raf GsA 25 GsC^(m)sC^(m)sC^(m)sAsAs GsC^(m)sTs human GsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1C^(m)SA

[0339] All nucleosides in bold are 2=-O-(methoxyethyl); subscript sindicates a phosphorothioate linkage; underlined nucleosides indicate2′-ara-(OH) modification. superscript m on C (Cm)indicates a 5-methyl-C.TABLE XIV Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-ara-(OMe) modifica- tions SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA-3′

[0340] All nucleosides in bold are 2=-O-(methoxyethyl); subscript Sindicates a phosphorothioate linkage; underlined nucleosides indicate2′-ara-(OMe) modification. superscript m on C (C^(m))indicates a5-methyl-C.

[0341] Procedure 2

[0342] Enzymatic Degradation of 2′-O-modified Oligonucleotides

[0343] Three oligonucleotides are synthesized incorporating themodifications shown in Table 2 below at the 3′-end. These modifiedoligonucleotides are subjected to snake venom phosphodiesterase action.

[0344] Oligonucleotides (30 nanomoles) are dissolved in 20 mL of buffercontaining 50 mM Tris-HCl pH 8.5, 14 mM MgCl₂, and 72 mM NaCl. To thissolution 0.1 units of snake-venom phosphodiesterase (Pharmacia,Piscataway, N.J.), 23 units of nuclease P1 (Gibco LBRL, Gaithersberg,Md.), and 24 units of calf intestinal phosphatase (Boehringer Mannheim,Indianapolis, Ind.) are added and the reaction mixture is incubated at37 C. for 100 hours. HPLC analysis is carried out using a Waters model715 automatic injector, model 600E pump, model 991 detector, and anAlltech (Alltech Associates, Inc., Deerfield, Ill.)nucleoside/nucleotide column (4.6×250 mm). All analyses are performed atroom temperature. The solvents used are A: water and B: acetonitrile.Analysis of the nucleoside composition is accomplished with thefollowing gradient: 0-5 min., 2% B (isocratic); 5-20 min., 2% B to 10% B(linear); 20-40 min., 10% B to 50% B. The integrated area per nanomoleis determined using nucleoside standards. Relative nucleoside ratios arecalculated by converting integrated areas to molar values and comparingall values to thymidine, which is set at its expected value for eacholigomer. TABLE XV Relative Nuclease Resistance of 2′-Modified ChimericOligonucleotides 5′-TTT TTT TTT TTT TTT T*T*T*T*-3′ SEQ ID NO:26(Uniform phosphodiester) T* = 2′-modified T -S-Me -Me -2′-ara-(F)-2′-ara-(OH) -2′-ara-(OMe)

[0345] Procedure 3

[0346] General Procedure for the Evaluation of Chimeric C3′-endo andC2′-endo Modified Oligonucleotides Targeted to Ha-ras

[0347] Different types of human tumors, including sarcomas,neuroblastomas, leukemias and lymphomas, contain active oncogenes of theras gene family. Ha-ras is a family of small molecular weight GTPaseswhose function is to regulate cellular proliferation and differentiationby transmitting signals resulting in constitutive activation of ras areassociated with a high percentage of diverse human cancers. Thus, rasrepresents an attractive target for anticancer therapeutic strategies.

[0348] SEQ ID NO: 27 (5′-TsCsCs GsTsCs AsTsCs GsCsTs CsCsTs CsAsGsGsG-3′) is a 20-base phosphorothioate oligodeoxynucleotide targeting theinitiation of translation region of human Ha-ras and it is a potentisotype-specific inhibitor of Ha-ras in cell culture based on screeningassays (IC₅₀=45 nm). Treatment of cells in vitro with SEQ ID NO: 27results in a rapid reduction of Ha-ras mRNA and protein synthesis andinhibition of proliferation of cells containing an activating Ha-rasmutation. When administered at doses of 25 mg/kg or lower by dailyintraperitoneal injection (IP), SEQ ID NO: 27 exhibits potent antitumoractivity in a variety of tumor xenograft models, whereas mismatchcontrols do not display antitumor activity. SEQ ID NO: 27 has been shownto be active against a variety of tumor types, including lung, breast,bladder, and pancreas in mouse xenograft studies (Cowsert, L. M.Anti-cancer drug design, 1997, 12, 359-371). A second-generation analogof SEQ ID NO: 27, where the 5′ and 3′ termini (“wings”) of the sequenceare modified with 2′-methoxyethyl (MOE) modification and the backbone iskept as phosphorothioate (Table XVI, SEQ ID NO: 27 (5′-TsCsCs GsTsCsAsTsCs GsCsTs CsCsTs CsAsGs GsG-3′)), exhibits IC₅₀ of 15 nm in cellculture assays. thus, a 3-fold improvement in efficacy is observed fromthis chimeric analog. Because of the improved nuclease resistance of the2′-MOE phosphorothioate, SEQ ID NO: 27 (5′-TsCsCs GsTsCs AsTsCs GsCsTsCsCsTs CsAsGs GsG-3′) increases the duration of antisense effect invitro. This will relate to frequency of administration of this drug tocancer patients. SEQ ID NO: 27 (5′-TsCsCs GsTsCs AsTsCs GsCsTs CsCsTsCsAsGs GsG-3′) is currently under evaluation in ras dependent tumormodels (Cowsert, L. M. Anti-cancer drug design, 1997, 12, 359-371). Theparent compound, SEQ ID NO: 27, is in Phase I clinical trials againstsolid tumors by systemic infusion.

[0349] Antisense oligonucleotides having the 2′-Me modification areprepared and tested in the aforementioned assays in the manner describedto determine activity.

[0350] Ha-ras Antisense Oligonucleotides with chimeric C3′-endo andC2′-endo modifications and Their Controls. TABLE XVI Ha-ras AntisenseOligonucleotides With chimeric C3′-endo and C2′-endo modifications andTheir Con- trols. SEQ ID Back- NO: Sequence bone 2′-Modif. Comments 275′-TsCsCs GsTsCs P = S 2′-H parent AsTsCs GsCsTs CsCsTs CsAsGs GsG-3′ 285′-TsCsAs GsTsAs P = S 2′-H mismatch AsTsAs GsGsCs CsCsAs CsAsTs GsG-3′29 5′-ToToCo GsTsCs AsTs P = O/ 2′-O-Moe Parent Cs GsCsTs CoCoTo CoAo P= S/ in wings Gapmer Go GoG-3′ P = O (Mixed Backbone) 27 5′-TsCsCsGsTsCs AsTs P = S 2′-O-MOE Parent Cs GsCsTs CsCsTs CsAs in wings Gapmeras Gs GsG-3′ uniform thioate 29 5′-ToCoAo GsTsAs AsTs P = O in wingsGapmer As GsCsCs GsCsCs GsCo (mixed Co CoCoAo CoAoTo GoG- Backbone) 3′28 5′-TsCsAs GsTsAs AsTs P = S 2′-O-MOE Control As GsCsCs GsCsCs inwings Gapmer as CsCsAs CsAsTs GsC-3′ uniform Thioate 27 5′-TsCsCsGsTsCs AsTs P = S 2′-O-MOE Control Cs GsCsTs CsCsTs CsAs in wings GapmerGs GsG-3′ with MOE control 28 5′-TsCsAs GsTsAs AsTs P = S 2′-O-MOEControl As GsCsCs GsCsCs CsCs in wings Gapmer As CsAsTs GsC-3′ with MOEControl

[0351] Procedure 7

[0352] In Vivo Nuclease Resistance

[0353] The in vivo Nuclease Resistance of chimeric C3′-endo and C2′-endomodified oligonucleotides is studied in mouse plasma and tissues (kidneyand liver). For this purpose, the C-raf oligonucleotide series SEQ IDNO: 30 are used and the following five oligonucleotides listed in theTable below will be evaluated for their relative nuclease resistance.TABLE XVII Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-S-Me) modi- fied oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothio- atelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 30 5′-ATG CAT TCT GCC P = S, (control) CCA AGGA-3′ 2′-Hrodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs P = O/2′-MOE/2′-S-Me/ GsCsCs CsCsAo AoGoGo P = S/ 2′-MOE A P = O 32 AsTsGsCsAsTs TsCsTs P = S 2′-MOE/2′-S-Me/ GsCsCs CsCsAs AsGsGs 2′-MOE A 33Ao*ToGo CoAsTs TsCsTs P = O/ In asterisk, 2′-5′ GsCsCs CsCsAo AoGoGo P= S/ linkage with 3′-O- *A P = O MOE; 2′-O-MOE/2′-S- Me/2′-O-MOE/2′-5′linkage with 3′-O- MOE in asterisk; 34 As*TsGs CsAsTs TsCsTs P = S Inasterisk, 2′-5′ GsCsCs CsCsAs AsGsGs linkage with *A 3′-O-MOE; 2′-O-MOE/2′-S-Me/2′-O- MOE/2′-5′ linkage with 3′-O-MOE in asterisk.

[0354] TABLE XVIII Study of in vivo Nuclease Resistance of chimericC3′-endo (2′-O-MOE) and C2′-endo (2′-Me) modified oligonucleotides withand without nuclease resi- stant caps (2′-5′-phosphate orphosphorothioate linkage with 3′-O-MOE in cap ends). SEQ ID Back- NO:Sequence bone Description 30 5′-ATG CAT TCT GCC CCA P = S, (control)AGGA-3′ 2′-H rodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs Gs P= O/ 2′-MOE/2′-Me/ CsCs CsCsAo AoGoGo A′ P = S/ 2′-MOE P = O 32 AsTsGsCsAsTs TsCsTs Gs P = S 2′-MOE/2′-Me/ CsCs CsCsAs AsGsGs A 2′-MOE   33Ao*ToGo CoAsTs TsCsTs Gs P = O/ In asterisk, 2′- CsCs CsCsAo AoGoGo *A P= S 5′ linkage with /P = O 3′-O-MOE; 2′-O- MOE/ 2′-Me/2′-O-MOE/2′-5′ link- age with 3′-O- MOE in asterisk;   34 As*TsGs CsAsTsTsCsTs Gs P = S In asterisk, 2′- CsCs CsCsAs AsGsGs *A 5′ linkage with3′-O-MOE; 2′-O- MOE/ 2′-Me/2′-O- MOE/2′-5′ link- age with 3′-O- MOE inasterisk;

[0355] TABLE XIX Study of in vivo Nuclease Resistance of chimericC3′-endo (2′-O-MOE) and C2′-endo (2′-ara-F) modi- fied oligonucleotideswith and without nuclease resistant caps (2′-5′-phosphate orphosphorothio- ate linkage with 3′-O-MOE in cap ends). SEQ ID Back- NO:Sequence bone Description 30 5′-ATG CAT TCT GCC CCA P = S, (control)AGGA-3′ 2′-H rodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs P = O/2′-MOE/2′-ara-F/ GsCsCs CsCsAo AoGoGo A P = S/ 2′-MOE P = O 32 AsTsGsCsAsTs TsCsTs P = S 2′-MOE/2′-ara- CsCsAs GsCsCs AsGsGs A F/2′-MOE 33Ao*ToGo CoAsTs TsCsTs P = O/ In asterisk, 2′- GsCsCs CsCsAo AoGoGo *A P= S/ 5′ linkage with P = O 3′-O-MOE; 2′-O-MOE/ 2′-ara-F/2′-O-MOE/2′-5′ link- age with 3′-O- MOE in asterisk; 34 As*TsGs CsAsTs TsCsTsP = S In asterisk, 2′- GsCsCs CsCsAs AsGsGs *A 5′ linkage with 3′-O-MOE;2′-O -MO 2′-ara-F/2′-O- MOE/2′-5′ link- age with 3′-O- MOE in asterisk;

[0356] TABLE XX Study of in vivo Nuclease Resistance of chimericC3′-endo (2′-O-MOE) and C2′-endo (2′-ara-OH) modi- fied oligonucleotideswith and without nuclease resistant caps (2′-5′-phosphate orphosphorothio- ate linkage with 3′-O-MOE in cap ends). SEQ ID Back- NO:Sequence bone Description 30 5′-ATG CAT TCT GCC CCA P = S, (control)AGGA-3′ 2′-H rodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs P = O/2′-MOE/2′-ara- GsCsCs CsCsAo AoGoGo A P = S/ OH/2′-MOE P = O 32 AsTsGsCsAsTs TsCsTs P = S 2′-MOE/2′-ara- GsCsCs CsCsAs AsGsGs A OH/2′-MOE 33Ao*ToGo CoAsTs TsCsTs P = O/ In asterisk, 2′- GsCsCs CsCsAo AoGoGo *A P= S/ 5′ linkage with P = O 3′-O-MOE; 2′-O- MOE/2′-ara-OH/ 2′-O-MOE/2′-5′ link- age with 3′-O- MOE in asterisk; 34 As*TsGs CsAsTs TsCsTsP = S In asterisk, 2′- GsCsCs CsCsAs AsGsGs *A 5′ linkage with 3′-O-MOE;2′-O- MOE/2′-ara-OH/ 2′-O-MOE/2′-5′ linkage with 3′- O-MOE in asterisk;

[0357] TABLE XXI Study of in vivo Nuclease Resistance of chimeric C3′-endo (2′-O-MOE) and C2′-endo (2′-ara-OMe) modi- fied oligonucleotideswith and without nuclease resistant caps (2′5′-phosphate orphosphorothioate linkage with 3′-O-MOE in cap ends). SEQ ID Back- NO:Sequence bone Description 30 5′-ATG CAT TCT GCC CCA P = S, (control) ro-AGG A-3′ 2′-H dent C-raf antisense oli- 31 AoToGo CoAsTs TsCsTs GsCsCs P= O/ go 2′-MOE/2′- CsCsAo AoGoGo Aa P = S/ ara-OMe/2′-MOE P = O 32AsTsGs CsAsTs TsCsTs GsCsCs P = S 2′-MOE/2′-ara- CsCsAs AsGsGs AOMe/2′-MOE 33 Ao*ToGo CoAsTs TsCsTs GsCsCs P = O/ In asterisk, CsCsAoAoGoGo *A P = S/ 2′-5′ linkage P = O with 3′-O-MOE; 2′-O-MOE/2′-ara-OMe/2′-O- MOE/2′-5′ link- age with 3′-O- MOE in aste- risk; 34As*TsGs CsAsTs TsCsTs GsCsCs P = S In asterisk, CsCsAs AsGsGs *A2′-5′ linkage with 3′-O-MOE; 2′-O-MOE/2′- ara-OMe/2′-O- MOE/2′-5′linkage with 3′-O-MOE in asterisk.

[0358] Procedure 8

[0359] Animal Studies for In Vivo Nuclease Resistance

[0360] For each oligonucleotide to be studied, 9 male BALB/c mice(Charles River, Wilmington, Mass.), weighing about 25 g are used (Crookeet al., J. Pharmacol. Exp. Ther., 1996, 277, 923). Following a 1-weekacclimation, the mice receive a single tail vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0. The final concentration of oligonucleotide in the dosingsolution is (5 mg/kg) for the PBS formulations. One retro-orbital bleed(either 0.25, 9.05, 2 or 4 post dose) and a terminal bleed (either 1, 3,8 or 24 h post dose) is collected from each group. The terminal bleed(approximately 0.6-0.8 mL) is collected by cardiac puncture followingketamine/xylazine anesthesia. The blood is transferred to an EDTA-coatedcollection tube and centrifuged to obtain plasma. At termination, theliver and kidneys will be collected from each mouse. Plasma and tissueshomogenates will be used for analysis for determination of intactoligonucleotide content by CGE. All samples are immediately frozen ondry ice after collection and stored at −80° C. until analysis.

[0361] Procedure 9

[0362] RNase H Studies with Chimeric C3′-endo and C2′-endo ModifiedOligonucleotides with and without Nuclease Resistant Caps

[0363]³²P Labeling of Oligonucleotides

[0364] The oligoribonucleotide (sense strand) was 5′-end labeled with³²P using [³²P]ATP, T4 polynucleotide kinase, and standard procedures(Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.G., Smith, J. A., and Struhl, K., in Current Protocols in MolecularBiology, John Wiley, New York (1989)). The labeled RNA was purified byelectrophoresis on 12% denaturing PAGE (Sambrook, J., Frisch, E. F., andManiatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Plainview (1989)). The specific activity of thelabeled oligonucleotide was approximately 6000 cpm/fmol.

[0365] Determination of RNase H Cleavage Patterns

[0366] Hybridization reactions were prepared in 120 μL of reactionbuffer [20 mM Tris-HC (pH 7.5), 20 mM KCl, 10 mM MgCl₂, 0.1 mM DTT]containing 750 nM antisense oligonucleotide, 500 nM senseoligoribonucleotide, and 100,000 cpm ³²P-labeled senseoligoribonucleotide. Reactions were heated at 90° C. for 5 min and 1unit of Inhibit-ACE was added. Samples were incubated overnight at 37°C. degrees. Hybridization reactions were incubated at 37° C. with1.5×10.8⁻⁸ mg of E. coli RNase H enzyme for initial rate determinationsand then quenched at specific time points. Samples were analyzed bytrichloroacetic acid (TCA) assay or by denaturing polyacrylamide gelelectrophoresis as previously described [Crooke, S. T., Lemonidis, K.M., Neilson, L., Griffey, R., Lesnik, E. A., and Monia, B. P., Kineticcharacteristics of Escherichia coli RNase H1: cleavage of variousantisense oligonucleotide-RNA duplexes, Biochem J, 312, 599 (1995);Lima, W. F. and Crooke, S. T., Biochemistry 36, 390-398, 1997].

[0367] Those skilled in the art will appreciate that numerous changesand modifications can be made to the preferred embodiments of theinvention and that such changes and modifications can be made withoutdeparting from the spirit of the invention. It is therefore intendedthat the appended claims cover all such equivalent variations as fallwithin the true spirit and scope of the invention.

What is claimed is:
 1. An oligonucleotide comprising a plurality ofnucleotides, wherein: a first portion of said plurality of nucleotideshave B-form conformational geometry and are joined together in acontinuous sequence, at least two of said nucleotides of said firstportion being ribonucleotides or arabinonucleotides; and a furtherportion of said plurality of nucleotides are ribonucleotide that haveA-form conformation geometry and are joined together in at least onecontinuous sequence.
 2. The oligonucleotide of claim 1 wherein eachnucleotide of said first portion, independently, is a 2′-SCH₃ribonucleotide, a 2′-NH₂ ribonucleotide, a 2′-NH(C₁-C₂ alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, a 2′-CF₃ribonucleotide, a 2′=CH₂ ribonucleotide, a 2′=CHF ribonucleotide, a2′=CF₂ ribonucleotide, a 2′-CH₃ ribonucleotide, a 2′-C₂H₅ribonucleotide, a 2′-CH═CH₂ ribonucleotide or a 2′-C≡CH ribonucleotide.3. The oligonucleotide of claim 1 wherein each of said nucleotides ofsaid first portion are joined together in said continuous sequence byphosphate, phosphorothioate, phosphorodithioate or boranophosphatelinkages.
 4. The oligonucleotide of claim 1 wherein each nucleotide ofsaid further portion, independently, is a 2′-fluoro nucleotide or anucleotide having a 2′-substituent having the formula I or II:

wherein E is C₁-C₁₀ alkyl, N(Q₁)(Q₂) or N═C(Q₁)(Q₂); each Q₁ and Q₂ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport, or Q₁ and Q₂, together, are joined in a nitrogen protectinggroup or a ring structure that can include at least one additionalheteroatom selected from N and O; R₃ is OX, SX, or N(X)₂; each X is,independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)Z, C(═O)N(H)Zor OC(═O)N(H)Z; Z is H or C₁-C₈ alkyl; L₁, L₂ and L₃ form a ring systemhaving from about 4 to about 7 carbon atoms or having from about 3 toabout 6 carbon atoms and 1 or 2 heteroatoms selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; Y is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q₁)(Q₂), O(Q₁),halo, S(Q₁), or CN; each q₁ is, independently, from 2 to 10; each q₂ is,independently, 0 or 1; m is 0, 1 or 2; p is from 1 to 10; and q₃ is from1 to 10 with the proviso that when p is 0, q₃ is greater than
 1. 5. Theoligonucleotide of claim 1 wherein each of said nucleotides of saidfurther portion, independently, is a 2′-F ribonucleotide, a 2′-O-(C₁-C₆alkyl) ribonucleotide, or a 2′-O-(C₁-C₆ substituted alkyl)ribonucleotide wherein the substitution is C₁-C₆ ether, C₁-C₆ thioether,amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.
 6. The oligonucleotideof claim 1 wherein all of said nucleotides of said further portion arejoined together in a continuous sequence by 3′-5′ phosphodiester, 2′-5′phosphodiester, phosphorothioate, Sp phosphorothioate, Rpphosphorothioate, phosphorodithioate, 3′-deoxy-3′-aminophosphoroamidate, 3′-methylenephosphonate, methylene(methylimino),dimethylhydrazino, amide 3, amide 4 or boranophosphate linkages.
 7. Theoligonucleotide of claim 1 wherein at least two of said nucleotides ofsaid further portion are joined together in a continuous sequence thatis positioned 3′ to said continuous sequence of said first portion ofsaid plurality of nucleotides.
 8. The oligonucleotide of claim 1 whereinat least two of said nucleotides of said further portion are joinedtogether in a continuous sequence that is positioned 5′ to saidcontinuous sequence of said first portion.
 9. The oligonucleotide ofclaim 1 wherein at least two of said nucleotides of said further portionare joined together in a continuous sequence that is positioned 3′ tosaid continuous sequence of said first portion and at least two of saidfurther portion are joined together in a continuous sequence that ispositioned 5′ to said continuous sequence of said first portion.
 10. Theoligonucleotide of claim 1 wherein each nucleotide of said firstportion, independently, is a 2′-SCH₃ ribonucleotide, a 2′-NH₂ribonucleotide, a 2′-NH(C₁-C₂ alkyl) ribonucleotide, a 2′-N(C₁-C₂alkyl)₂ ribonucleotide, a 2′=CH₂ ribonucleotide, a 2′-CH₃ribonucleotide, a 2′-C₂H₅ ribonucleotide, a 2′-CH═CH₂ ribonucleotide ora 2′-C≡CH ribonucleotide.
 11. The oligonucleotide of claim 1 whereineach nucleotide of said first portion, independently, is a 2′-SCH₃ribonucleotide, a 2′-NH₂ ribonucleotide a 2′-NH(C₁-C₂ alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ ribonucleotide or a 2′-CH₃ribonucleotide.
 12. The oligonucleotide of claim 1 wherein eachnucleotide of said first portion, independently, is a 2′-SCH₃ribonucleotide, a 2′-NH₂ ribonucleotide or a 2′-CH₃ ribonucleotide. 13.The oligonucleotide of claim 1 wherein each nucleotide of said firstportion is a 2′-SCH₃ ribonucleotide.
 14. The oligonucleotide of claim 1wherein each nucleotide of said first portion, independently, is a 2′-CNarabinonucleotide, a 2′-F arabinonucleotide, a 2′-Cl arabinonucleotide,a 2′-Br arabinonucleotide, a 2′-N₃ arabinonucleotide, a 2′-OHarabinonucleotide, a 2′-O—CH₃ arabinonucleotide or a 2′-dehydro-2′-CH₃arabinonucleotide.
 15. The oligonucleotide of claim 1 wherein eachnucleotide of said first portion, independently, is a 2′-Farabinonucleotide, a 2′-OH arabinonucleotide or a 2′-O—CH₃arabinonucleotide.
 16. The oligonucleotide of claim 1 wherein eachnucleotide of said first portion, independently, is a 2′-Farabinonucleotide or a 2′-OH arabinonucleotide.
 17. The oligonucleotideof claim 1 wherein each nucleotide of said first portion is a 2′-Farabinonucleotide.
 18. The oligonucleotide of claim 1 wherein eachnucleotide of said first portion, independently, is a 2′-SCH₃ribonucleotide, a 2′-NH₂ ribonucleotide a 2′-NH(C₁-C₂ alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, a 2′-CH₃ribonucleotide, a 2′-CH═CH₂ ribonucleotide or a 2′-C≡CH ribonucleotide;and each nucleotide of said further portion, independently, is a 2′-Fribonucleotide, a 2′-O—(C₁-C₆ alkyl) ribonucleotide, or a 2′-O—(C₁-C₆substituted alkyl) ribonucleotide wherein the substitution is C₁-C₆ether, C₁-C₆ thioether, amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆alkyl)₂.
 19. The oligonucleotide of claim 1 wherein each nucleotide ofsaid first portion, independently, is a 2′-CN arabinonucleotide, a 2′-Farabinonucleotide, a 2′-Cl arabinonucleotide, a 2′-Br arabinonucleotide,a 2′-N₃ arabinonucleotide, a 2′-OH arabinonucleotide, a 2′-O—CH₃arabinonucleotide or a 2′-dehydro-2′-CH₃ arabinonucleotide; and eachnucleotide of said further portion, independently, is a 2′-Fribonucleotide, a 2′-O—(C₁-C₆ alkyl) ribonucleotide, or a 2′-O—(C₁-C₆substituted alkyl) ribonucleotide wherein the substitution is C₁-C₆ether, C₁-C₆ thioether, amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆alkyl)₂.
 20. The oligonucleotide of claim 1 wherein each nucleotide ofsaid first portion, independently, is a 2′-F arabinonucleotide or a2′-OH arabinonucleotide; and each nucleotide of said further portion isa 2′-O—(C₁-C₆ substituted alkyl) ribonucleotide wherein the substitutionis C₁-C₆ ether, C₁-C₆ thioether, amino, amino(C₁-C₆ alkyl) oramino(C₁-C₆ alkyl)₂.
 21. The oligonucleotide of claim 1 wherein saidfurther portion comprises at least two nucleotides joined together in acontinuous sequence that is positioned at the 3′ terminus end of saidoligonucleotide.
 22. The oligonucleotide of claim 1 wherein said furtherportion comprises at least two nucleotides joined together in acontinuous sequence that is positioned at the 5′ terminus of saidoligonucleotide.
 23. The oligonucleotide of claim 1 wherein said furtherportion comprises at least two nucleotides joined together in acontinuous sequence that is positions at the 3′ terminus of saidoligonucleotide; and at least two nucleotides joined together in acontinuous sequence that is positions at the 5′ terminus of saidoligonucleotide.
 24. The oligonucleotide of claim 21 wherein said atleast two nucleotides joined together comprise nucleotides joinedtogether by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonatelinkage, a Sp phosphorothioate linkage, a methylene(methylimino)linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-aminophosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage. 25.The oligonucleotide of claim 24 wherein said two nucleotides are joinedtogether by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonatelinkage, a Sp phosphorothioate linkage or a methylene(methylimino)linkage.
 26. The oligonucleotide of claim 22 wherein said at least twonucleotides joined together comprise nucleotides joined together by a2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Spphosphorothioate linkage, a methylene(methylimino) linkage, adimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage,an amide 3 linkage or an amide 4 linkage.
 27. The oligonucleotide ofclaim 26 wherein said two nucleotides are joined together by a 2′-5′phosphodiester linkage, a 3′-methylenephosphonate linkage, a Spphosphorothioate linkage or a methylene(methylimino) linkage.
 28. Theoligonucleotide of claim 23 wherein said at least two nucleotides joinedtogether and positioned at said 3′ terminus comprise nucleotides joinedtogether by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonatelinkage, a Sp phosphorothioate linkage, a methylene(methylimino)linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-aminophosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage; andwherein said at least two nucleotides joined together and positioned atsaid 5′ terminus comprise nucleotides joined together by a 2′-5′phosphodiester linkage, a 3′-methylenephosphonate linkage, a Spphosphorothioate linkage, a methylene(methylimino) linkage, adimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage,an amide 3 linkage or an amide 4 linkage.
 29. The oligonucleotide ofclaim 28 wherein said two nucleotides joined together at said 3′terminus and said two nucleotides joined together at said 5′ terminusare, independently, joined together by 2′-5′ phosphodiester linkages,3′-methylenephosphonate linkages, Sp phosphorothioate linkages ormethylene(methylimino) linkages.
 30. The oligonucleotide of claim 21wherein at least one of said two nucleotides joined together is a2′-alkylamino substituted nucleotide.
 31. The oligonucleotide of claim22 wherein at least one of said two nucleotides joined together is a2′-alkylamino substituted nucleotide.
 32. The oligonucleotide of claim23 wherein at least one of said two nucleotides joined together at said3′ terminus is a 2′-alkylamino substituted nucleotide, and wherein atleast one of said two nucleotides joined together at said 5′ terminus isa 2′-alkylamino substituted nucleotide.
 33. An oligonucleotidecomprising a plurality of linked nucleotides, wherein: at least one ofsaid nucleotides has a C3′ endo type pucker; and at least two of saidplurality of nucleotides are joined together in a continuous sequenceand have a C2′ endo type pucker or an O4′ endo type pucker, providedthat said nucleotides are not 2′-deoxy-erythro-pentofuranosylnucleotides.
 34. The oligonucleotide of claim 33 wherein saidnucleotides having said C3′ endo type pucker are joined together in acontinuous sequence that is positioned 3′ to said continuous sequence ofnucleotides having said C2′ endo type pucker or O4′ endo type pucker.35. The oligonucleotide of claim 33 wherein said nucleotides having saidC3′ endo type pucker are joined together in a continuous sequence thatis positioned 5′ to said continuous sequence of nucleotides having saidC2′ endo type pucker or O4′ endo type pucker.
 36. The oligonucleotide ofclaim 33 wherein at least two of said nucleotides having said C3′ endotype pucker are joined together in a continuous sequence that ispositioned 3′ to said continuous sequence of said nucleotides havingsaid C2′ endo type pucker or O4′ endo type pucker; and at least two ofsaid nucleotides having said C3′ endo type pucker are joined together ina continuous sequence that is positioned 5′ to said continuous sequenceof said nucleotides having said C2′ endo type pucker or O4′ endo typepucker.